Methods for identifying and isolating cells expressing a polypeptide

ABSTRACT

The invention relates to novel polypeptides and cells comprising the polypeptides. The polypeptides and cells are used in methods to identify and/or isolate cells producing a protein with specific biological functions. In particular, the methods may be used for identifying, selecting, and isolating cells producing antigen-specific monoclonal antibodies.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.13/025,733, filed Feb. 11, 2011, now allowed, which claims the prioritybenefit of U.S. Provisional Application No. 61/304,251, filed Feb. 12,2010 and U.S. Provisional Application No. 61/437,889, filed Jan. 31,2011, each of which is hereby incorporated by reference herein in itsentirety.

FIELD OF THE INVENTION

The field of this invention generally relates to novel polypeptides andcells comprising the polypeptides. The invention also relates to usingthe polypeptides in methods to identify and/or isolate cells expressingthe polypeptides. The methods may be used for identifying and isolatingcells producing antigen-specific monoclonal antibodies.

BACKGROUND OF THE INVENTION

Since the development of monoclonal antibody technology in the 1970s,monoclonal antibodies have become an increasingly important class oftherapeutic agents. Hybridoma technology is still the most commonly usedmethod for producing monoclonal antibodies. The monoclonal antibodiesare secreted from hybridoma cells that are created by fusing normalantibody producing B-cells with immortal myeloma cells or other immortalcells. The process of monoclonal antibody development usually involvesseveral cycles of screening supernatants to identify a hybridomaproducing an antibody that binds to the antigen of interest.

The identification of hybridomas that produce monoclonal antibodies toan antigen of interest is typically accomplished by ELISA screening.Supernatant produced by pools of random hybridoma clones from ahybridoma library can be screened. This method has limitations, becauseit must be followed by limiting dilution of the positive pool(s) toisolate individual clones and then all clones need to be rescreened. Insome cases, the hybridoma library is cloned by limiting dilution as afirst step producing a very large number of individual clones to screen.Any method that includes a limiting dilution step is problematic,because it is time consuming and very labor intensive. Furthermore, insome circumstances the desired clone may represent an extremely lowpercentage of the hybridoma library, making the identification of therare clone difficult. In addition, the ELISA screening approachidentifies binding activity to a single antigen. To determine if amonoclonal antibody binds to more than one antigen, multiple successiveELISA screenings must be undertaken with the different individualantigens.

It would be advantageous if the cell producing an antibody retained theantibody in a form (e.g., at the surface of the cell) that would allowthe antibody-producing cell itself to be directly identified. Thisstrategy is one of the reasons phage display technology has been sosuccessful. In fact, normal B-cells make membrane-bound immunoglobulinand this molecule is a core component of the B-cell receptor complexthat signals in response to binding with antigen. The presence of nativemembrane-bound antibody has previously been used in an attempt todirectly isolate hybridomas (see, Parks et al. 1979, PNAS,76:1962-1966). However, the extremely low levels of membrane-boundantibody made this method of limited use. Several other techniques havebeen developed to further this goal. One method is the “secretioncapture report web” (SCRW) which encapsulates cells in biotinylatedagarose microdroplets and then successively incubates the dropsuspension with avidin and biotinylated anti-mouse IgG. Avidin serves asa bridge between the biotinylated agarose and the biotinylatedanti-mouse antibody to form capture sites within the drops to trapantibody being secreted by the cell. These antibody-containingmicrodroplets can be screened for the ability to bind a reporter (e.g.,a fluorescent-tagged antigen) and the droplets can be isolated by flowcytometry. (See, Kenney et al., 1995, Nature Biotechnology 8:787-90;Gray et al., 1995, J. Immunol. Methods 182:155-63.) Other methods arebased upon the ability to transiently capture a secreted protein orantibody on the surface of a cell. The “captured” protein or antibodycan be detected on the cell surface by binding of a reporter molecule(e.g., a fluorescent-tagged antigen) and isolated, for example, by flowcytometry (see, e.g., U.S. Pat. Nos. 6,919,183 and 7,166,423; U.S.Patent App. No. 2010/0009866).

Each of these techniques has limitations. The agarose microdroplettechnique is technically difficult and requires special equipment togenerate the agarose microdroplets. In addition cells can be sensitiveto the encapsulation process. The cell surface capture methods do notfully discriminate between the antibody produced by the hybridoma cellof interest and antibodies produced by other hybridoma cells. Diffusionof the antibody or protein of interest between neighboring cells can beproblematic. For example, an antibody can dissociate from the capturemolecule on the cell that produced it and diffuse to and be “captured”by a cell producing a different antibody. Thus in some cases, themethods require a high viscosity medium to reduce diffusion of theprotein or antibody away from the expressing cell. Further, not all ofthe antibody produced by a hybridoma is actually captured on the cellsurface and this excess antibody is secreted into the medium where it isreadily available to bind to the capture molecule on other randomhybridoma cells. Accordingly, new and/or improved methods foridentifying and selecting cells producing antigen-specific monoclonalantibodies are needed.

SUMMARY OF THE INVENTION

The present invention describes novel polypeptides and cells comprisingthe polypeptides, as well as methods of using the polypeptides, cellsand cells libraries to identify and/or select cells producingpolypeptides. In particular, the methods may be used to identify andisolate cells producing antigen-specific monoclonal antibodies. Theinvention provides an approach wherein a membrane-bound heterodimericmolecule comprising a single antigen-binding site is expressed on thesurface of the cell. The single antigen-binding site is representativeof the binding specificity of the antibody produced by the cell. Theheterodimeric molecule does not “bind” secreted antibody, so there arelimited or no problems with antibody produced by one cell being bound orpresented on the surface of another cell. The method and constructs asdescribed herein are referred to as “Membrane-MAb” or “Membrane-MAbtechnique” and “Membrane-MAb constructs”. The novel polypeptideconstructs comprise a polypeptide comprising a dimerization domain and atransmembrane region from an immunoglobulin or non-immunoglobulinprotein. In some embodiments, the novel polypeptide constructs comprisean immunoglobulin heavy chain constant region comprising CH2 and CH3domains and a transmembrane region from an immunoglobulin ornon-immunoglobulin protein. In some embodiments, the novel polypeptideconstructs comprise an immunoglobulin heavy chain constant regioncomprising CH2 and CH3 domains and a GPI(glycosylphosphatidylinositol)-membrane anchor. When a cell expressesboth the polypeptide and, for example, an immunoglobulin heavy chain,the polypeptides associate to produce a heterodimeric moleculecomprising a monovalent antibody that is expressed on the surface of thecell. The heterodimeric molecule is not an antibody-binding protein andas such it does not bind or capture secreted antibody. A non-limitingexample of the Membrane-MAb strategy is depicted in FIG. 1C. This iscompared to a traditional hybridoma technique (FIG. 1A) and an exampleof a surface capture method (FIG. 1B).

In one aspect, the invention provides a polypeptide that is able to forma heterodimeric molecule with a second polypeptide, wherein first thepolypeptide is membrane-bound and the heterodimeric molecule isexpressed on the surface of a cell. In some embodiments, the polypeptidecomprises (a) an extracellular portion comprising a dimerization domain,and (b) a transmembrane portion. In some embodiments, the dimerizationdomain may include, but is not limited to, a Fc region, animmunoglobulin constant region, a leucine zipper, or an isoleucinezipper. In some embodiments, the dimerization domain may be taken from areceptor, an integrin, or any molecule that normally forms a dimeric ormulitmeric structure. The dimerization domain may be taken from, forexample, immunoglobulin, LFA-1, GPIIIb/IIIa), nerve growth factor (NGF),neurotrophin-3 (NT-3), interleukin-8 (IL-8), IL-8 receptor, vascularendothelial growth factor (VEGF), brain-derived neurotrophic factor(BDNF), Fos, Jun, NFkB, Ras, Raf, CD4, Bcl-2, Myc and Met.

In some embodiments, the polypeptide comprises (a) an extracellularportion comprising an immunoglobulin heavy chain constant region, and(b) a transmembrane portion. In some embodiments, the polypeptidecomprises: (a) an extracellular portion comprising an immunoglobulinheavy chain constant region comprising CH2 and CH3 domains; and (b) anon-immunoglobulin transmembrane portion. In some embodiments, theimmunoglobulin heavy chain constant region comprises at least a portionof a hinge region, CH2 and CH3 domains. In some embodiments, theimmunoglobulin heavy chain constant region comprises a Fc region. Insome embodiments, the immunoglobulin heavy chain constant region is froman IgG, IgA, IgD, IgE, or IgM antibody or a subtype thereof. In certainembodiments, the immunoglobulin heavy chain constant region is from anIgG1 or an IgG2 antibody. In some embodiments, the immunoglobulin heavychain constant region is a mouse immunoglobulin heavy chain constantregion. In some embodiments, the immunoglobulin heavy chain constantregion is a human immunoglobulin heavy chain constant region. In someembodiments, the immunoglobulin heavy chain constant region comprisesSEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:8.

In some embodiments, the polypeptides of the present invention comprisea transmembrane portion. In some embodiments, the transmembrane portioncomprises at least a portion of a transmembrane domain from animmunoglobulin or a non-immunoglobulin protein. In some embodiments, thetransmembrane portion is from a human protein. In certain embodiments,the transmembrane portion is from a mouse protein. In some embodiments,the transmembrane portion is from a B-cell immunoglobulin protein. Insome embodiments, the transmembrane portion is from a mouse B-cellimmunoglobulin protein. In some embodiments, the transmembrane portionis from a human B-cell immunoglobulin protein. In some embodiments, thetransmembrane portion is from a protein selected from the groupconsisting of: CD4, CD8, Class I MHC, Class II MHC, CD19, T-cellreceptor α and β chains, CD3, zeta chain, ICAM1 (CD54), ICAM2, ICAM3,ICAM4, ICAM5, CD28, CD79a, CD79b, and CD2. In certain embodiments, thetransmembrane portion is from a CD4 protein. In certain embodiments, thetransmembrane portion is from a mouse CD4 protein. In certainembodiments, the transmembrane portion is from a human CD4 protein. Incertain embodiments, the transmembrane portion comprises SEQ ID NO:13 orSEQ ID NO:16. In some embodiments, the transmembrane portion furthercomprises an intracellular domain (ICD). In some embodiments, thetransmembrane portion comprises SEQ ID NO:14, SEQ ID NO:15, or SEQ IDNO:17.

In some embodiments, the polypeptides of the present invention comprisea GPI-membrane anchor. In some embodiments, the GPI-membrane anchor isobtained from a protein selected from the group consisting of CD52,CD55, CD58, and CD59.

In some embodiments, the polypeptides of the present invention comprisea detection or “reporter” molecule. In some embodiments, the detectionor reporter molecule is a fluorescent protein, a bioluminescent protein,or a variant thereof. In certain embodiments, the detection or reportermolecule is green fluorescent protein (GFP) or a variant thereof.

Thus in some embodiments, the polypeptides of the present inventioncomprise an IgG CH2CH3 region and a transmembrane domain. In certainembodiments, the polypeptides comprise an IgG CH2CH3 constant region, atransmembrane domain, and GFP.

In some embodiments, the polypeptides of the present invention aremembrane-bound and the immunoglobulin heavy chain constant region isexpressed on the surface of the cell. In some embodiments, thepolypeptide does not comprise an immunoglobulin heavy chain variableregion. In certain embodiments, the polypeptide does not comprise anantigen-binding site. In some embodiments, the polypeptide is able toform a heterodimeric molecule with a second polypeptide. In certainembodiments, the second polypeptide comprises an immunoglobulin Fcregion. In certain embodiments, the second polypeptide comprises animmunoglobulin heavy chain. In some embodiments, the second polypeptidefurther comprises an immunoglobulin light chain. In some embodiments,the second polypeptide comprises a single chain immunoglobulin with bothan immunoglobulin heavy chain and an immunoglobulin light chain. In someembodiments, the polypeptide is able to form at least one disulfide bondwith a second polypeptide.

In some embodiments, the polypeptide of the present invention comprisesSEQ ID NO:10, SEQ ID NO:12, or SEQ ID NO:28. In some embodiments, thepolypeptide of the present invention comprises SEQ ID NO:22, SEQ IDNO:23, or SEQ ID NO:26. In certain embodiments, the polypeptide of thepresent invention comprises SEQ ID NO:30, SEQ ID NO:31, or SEQ ID NO:32.In some embodiments, the polypeptide is encoded by a sequence comprisingSEQ ID NO:9, SEQ ID NO:11, or SEQ ID NO:29. In some embodiments, thepolypeptide is encoded by a sequence comprising SEQ ID NO:24, SEQ IDNO:25, or SEQ ID NO:27. In some embodiments, a host cell expresses anyof the polypeptides described herein. In some embodiments, a host cellproduces any of the polypeptides described herein.

In another aspect, the invention provides a heterodimeric polypeptidemolecule. In some embodiments, the heterodimeric polypeptide moleculecomprises (a) a first polypeptide comprising (i) an extracellularportion comprising a dimerization domain and (ii) a transmembraneportion, and (b) a second polypeptide comprising a dimerization domain.In some embodiments, the heterodimeric molecule comprises a firstpolypeptide comprising an immunoglobulin heavy chain. In someembodiments, the heterodimeric molecule comprises a second polypeptidecomprising an immunoglobulin heavy chain. In some embodiments, thesecond polypeptide further comprises an immunoglobulin light chain. Insome embodiments, the second polypeptide comprises a single chainimmunoglobulin with both an immunoglobulin heavy chain and animmunoglobulin light chain. In some embodiments, the first polypeptideforms at least one disulfide bond with the second polypeptide.

In another aspect, the invention provides an antibody moleculecomprising any of the polypeptides described herein. In someembodiments, the antibody molecule is a heterodimeric molecule. Incertain embodiments, the antibody molecule further comprises: (a) animmunoglobulin heavy chain, and (b) an immunoglobulin light chain. Insome embodiments, the antibody molecule further comprises a single chainimmunoglobulin with an immunoglobulin heavy chain and an immunoglobulinlight chain. In some embodiments, the polypeptide of the antibodymolecule forms at least one disulfide bond with the Fc region of animmunoglobulin heavy chain-light chain pair. In some embodiments, theantibody molecule comprises a single antigen-binding site (e.g., ismonovalent).

In one aspect, the invention provides a polynucleotide that encodes anyof the polypeptides described herein. In some embodiments, thepolynucleotide comprises SEQ ID NO:9, SEQ ID NO:11, or SEQ ID NO:29. Insome embodiments, the polynucleotide comprises SEQ ID NO:24, SEQ IDNO:25, or SEQ ID NO:27. In some embodiments, a vector comprises thepolynucleotide. In some embodiments, a host cell comprises thepolynucleotide or vector. In some embodiments, a host cell comprises anyof the polypeptides or antibody molecules described herein.

In one aspect, the invention provides host cells and methods ofproducing a host cell comprising the polypeptides described herein. Insome embodiments, a method of producing a host cell comprisestransfecting a cell with a polynucleotide that encodes a polypeptidecomprising: (a) an extracellular portion comprising an immunoglobulinheavy chain constant region, and (b) a transmembrane portion. In someembodiments, the transfected cell expresses the polypeptide. In someembodiments, the cell is transiently transfected. In other embodiments,the cell is stably transfected. In some embodiments, the method furthercomprises detecting expression of the polypeptide. In some embodiments,the method further comprises detecting expression of the polypeptide onthe surface of the cell. In other embodiments, the method furthercomprises isolating a cell that expresses the polypeptide on the surfaceof the cell. In some embodiments, the cell is a mammalian cell (e.g., ahuman or mouse cell). In some embodiments, the cell is a fusion partnercell line.

In some embodiments, the invention provides host cells produced by themethods described herein. In some embodiments, the cells comprise apolynucleotide that encodes any of the polypeptides described herein. Insome embodiments, the cells comprise any of the polypeptides describedherein. In certain embodiments, the cells express a polypeptide, whereinthe polypeptide comprises: (a) an extracellular portion comprising animmunoglobulin heavy chain constant region, and (b) a transmembraneportion.

In another aspect, the cells of the present invention may be used toproduce membrane-bound heterodimeric molecules. In some embodiments, acell comprises: (a) a polynucleotide that encodes a membrane-boundpolypeptide comprising: (i) an extracellular portion comprising animmunoglobulin heavy chain constant region, and (ii) a transmembraneportion; and (b) at least one additional polynucleotide that encodes atleast one additional polypeptide. In some embodiments, the additionalpolypeptide comprises an immunoglobulin heavy chain constant regioncomprising CH2 and CH3 domains. In some embodiments, the additionalpolypeptide comprises a Fc domain. In some embodiments, the additionalpolypeptide comprises an immunoglobulin heavy chain and/or light chain.In some embodiments, the additional polypeptide comprises an antibody.In some embodiments, the additional polypeptide comprises a single chainantibody. In some embodiments, the at least one additional polypeptidecomprises randomized polypeptides. In some embodiments, the at least oneadditional polypeptide comprises mutagenized polypeptides. In someembodiments, the at least one additional polypeptide comprises a libraryof polypeptides. In some embodiments, the additional polypeptides aresecreted from the cell. In other embodiments, the membrane-boundpolypeptide forms at least one disulfide bond with the additionalpolypeptide to form a membrane-bound heterodimeric molecule.

In one aspect, the invention provides methods of producing a hybridomacell that expresses a membrane-bound heterodimeric molecule on thesurface of the cell. In some embodiments, a method of producing ahybridoma cell comprises fusing cells with an antibody-producing cell,wherein the cells comprise a polynucleotide that encodes amembrane-bound polypeptide comprising: (a) an extracellular portioncomprising an immunoglobulin heavy chain constant region, and (b) atransmembrane portion. In some embodiments, the fused hybridoma cellsexpress a heterodimeric antibody molecule on the surface of the cells.In some embodiments, the antibody-producing cell is a population ofantibody-producing cells. In some embodiments, the antibody-producingcell is from a naive animal. In some embodiments, the antibody-producingcell is from an immunized animal. In some embodiments, theantibody-producing cell includes, but is not limited to, a B-cell, aplasma cell, a hybridoma, a myeloma, and a recombinant cell. In someembodiments, the antibody-producing cell comprises a plurality ofpolynucleotides. In some embodiments, the plurality of polynucleotidesencodes a plurality of polypeptides. In some embodiments, the pluralityof polypeptides comprises immunoglobulin heavy chain constant regions.In some embodiments, the plurality of polypeptides comprisesimmunoglobulin heavy chains, and/or immunoglobulin light chains. In someembodiments, the plurality of polypeptides comprises a single chainimmunoglobulin with an immunoglobulin heavy chain and an immunoglobulinlight chain. In some embodiments, the plurality of polypeptidescomprises a randomized polypeptide library. In some embodiments, theplurality of polynucleotides comprises a DNA library. In someembodiments, the DNA library is generated from cells of a naïve animal.In some embodiments, the DNA library is generated from cells of animmunized animal. In some embodiments, the DNA library is a cDNAlibrary. In some embodiments, the fused cells comprise a population ofhybridoma cells that express a plurality of heterodimeric antibodymolecules.

In one aspect, the invention provides a hybridoma or hybridoma librarymade by any of the methods described herein.

In another aspect, the invention provides cell libraries and methods ofproducing cell libraries comprising the polypeptides described herein.In some embodiments, a method of producing a cell library comprisestransfecting cells with a plurality of polynucleotides, wherein thecells comprise a polynucleotide that encodes a polypeptide comprising(a) an extracellular portion comprising an immunoglobulin heavy chainconstant region, and (b) a transmembrane portion. In some embodiments,the transfected cells express a heterodimeric molecule on the surface ofa plurality of the transfected cells. In some embodiments, the pluralityof polynucleotides encodes a plurality of polypeptides. In someembodiments, each polypeptide of the plurality of polypeptides comprisesan immunoglobulin Fc region. In some embodiments, the plurality ofpolypeptides comprises a plurality of randomized polypeptides. In someembodiments, each polypeptide of the plurality of polypeptidescomprises: (a) an immunoglobulin Fc region, and (b) a randomizedpolypeptide. In other embodiments, the plurality of polypeptidescomprises immunoglobulin heavy chains, and/or immunoglobulin lightchains. In other embodiments, the plurality of polypeptides comprises asingle chain immunoglobulin with an immunoglobulin heavy chain and animmunoglobulin light chain. In some embodiments, the plurality ofpolynucleotides comprises a DNA library. In some embodiments, the DNAlibrary is generated from cells of a naïve animal. In some embodiments,the DNA library is generated from cells of an immunized animal. In otherembodiments, the DNA library encodes a plurality of randomizedpolypeptides. In other embodiments, the DNA library encodes a pluralityof polypeptides, wherein each polypeptide comprises: (a) animmunoglobulin Fc region, and (b) a randomized polypeptide.

In one aspect, the invention provides a cell library made by any of themethods described herein.

In another aspect, the invention provides methods of identifying a cellthat is producing a specific antibody. In some embodiments, a method ofidentifying a cell that produces a specific antibody comprises fusingcells with an antibody-producing cell to produce a population ofhybridoma cells, wherein the cells comprise a polypeptide comprising (a)an extracellular portion comprising an immunoglobulin heavy chainconstant region, and (b) a transmembrane portion. In some embodiments,the hybridoma cells express a heterodimeric molecule on the surface ofthe cells. In some embodiments, the method comprises contacting thepopulation of hybridoma cells with a detection molecule (e.g., a targetof interest). In some embodiments, the method comprises identifying thehybridoma cells that are bound by the detection molecule. In someembodiments, the method comprises isolating the cells that are bound bythe detection molecule. In some embodiments, the antibody-producingcells are from a naïve animal. In some embodiments, theantibody-producing cells are from an immunized animal. In someembodiments, the antibody-producing cells are human cells. In someembodiments, the antibody-producing cells are mouse cells. In certainembodiments, the antibody-producing cell is a B-cell, a plasma cell, ahybridoma, a myeloma, or a recombinant cell. In some embodiments, theantibody-producing cell comprises a plurality of polypeptides. In someembodiments, the antibody-producing cell comprises a plurality ofpolynucleotides. In some embodiments, the plurality of polynucleotidesencodes a plurality of polypeptides comprising immunoglobulin heavychains, and/or immunoglobulin light chains. In some embodiments, theplurality of polynucleotides encodes a plurality of polypeptidescomprising a single chain immunoglobulin with an immunoglobulin heavychain and an immunoglobulin light chain. In other embodiments, theplurality of polynucleotides comprises a DNA library. In someembodiments, the DNA library is generated from cells of a naïve animal.In some embodiments, the DNA library is generated from cells of animmunized animal.

In some embodiments, the antibody made by the antibody-producing cellsis a recombinant antibody, a monoclonal antibody, a chimeric antibody, ahumanized antibody, a human antibody, or an antibody fragment. In someembodiments, the antibody made by the antibody-producing cell is an IgA,IgD, IgE, IgG or IgM antibody or a subtype thereof.

In some embodiments, the detection molecule (e.g., a target of interest)is a protein or a fragment thereof. In some embodiments, the detectionmolecule is an antigen of interest. In some embodiments, the detectionmolecule is labeled. In certain embodiments, the cells bound by thedetection molecule are identified by flow cytometry. In someembodiments, the cells bound by the detection molecule are isolated byfluorescence-activated cell sorting (FACS).

In some embodiments, the method of identifying a cell that produces aspecific antibody comprises transfecting cells with at least onepolynucleotide, wherein the cells comprise a polypeptide comprising (a)an extracellular portion comprising an immunoglobulin heavy chainconstant region, and (b) a transmembrane portion. In some embodiments,the transfected cells express a heterodimeric molecule on the surface ofthe cells. In some embodiments, the method comprises contacting thetransfected cells with a detection molecule (e.g., a target ofinterest). In some embodiments, the method comprises identifying thecells that are bound by the detection molecule. In some embodiments, themethod comprises isolating the cells that are bound by the detectionmolecule. In some embodiments, the at least one polynucleotide comprisesa plurality of polynucleotides. In some embodiments, the plurality ofpolynucleotides encodes a plurality of polypeptides comprisingimmunoglobulin heavy chains, and/or immunoglobulin light chains. Inother embodiments, the plurality of polynucleotides comprises a DNAlibrary. In some embodiments, the DNA library is generated from cells ofa naïve animal. In some embodiments, the DNA library is generated fromcells of an immunized animal.

In some embodiments, the at least one polynucleotide encodes for apolypeptide that is a recombinant antibody, a monoclonal antibody, achimeric antibody, a humanized antibody, a human antibody, or anantibody fragment. In some embodiments, the at least one polynucleotideencodes for a polypeptide that is an IgA, IgD, IgE, IgG or IgM antibodyor a sub-type thereof.

In some embodiments, the detection molecule (e.g., a target of interest)is a protein or fragment thereof. In some embodiments, the detectionmolecule is an antigen of interest. In some embodiments, the detectionmolecule is labeled. In certain embodiments, the cells bound by thedetection molecule are identified by flow cytometry. In someembodiments, the cells bound by the detection molecule are isolated byFACS.

In some embodiments, the method of identifying a cell that produces aspecific antibody comprises transfecting a cell library with apolynucleotide encoding a polypeptide comprising (a) an extracellularportion comprising an immunoglobulin heavy chain constant region, and(b) a transmembrane portion, wherein the cell library comprisesantibody-producing cells. In some embodiments, the transfected cellsexpress a heterodimeric molecule on the surface of the cells. In someembodiments, the method comprises contacting the transfected cells witha detection molecule (e.g., a target of interest). In some embodiments,the method comprises identifying the cells that are bound by thedetection molecule. In some embodiments, the method comprises isolatingthe cells that are bound by the detection molecule. In some embodiments,the cell library is a hybridoma library. In some embodiments, the celllibrary comprises cells are from a naïve animal. In some embodiments,the cell library comprises cells are from an immunized animal. In someembodiments, the cell library comprises human cells. In someembodiments, the cell library comprises mouse cells. In certainembodiments, the cell library comprises B-cells, plasma cells,hybridomas, myelomas, or recombinant cells. In some embodiments, thecell library comprises a plurality of polypeptides. In some embodiments,the cell library comprises a plurality of polynucleotides. In someembodiments, the plurality of polynucleotides encodes a plurality ofpolypeptides comprising immunoglobulin heavy chains, and/orimmunoglobulin light chains. In some embodiments, the plurality ofpolynucleotides encodes a plurality of polypeptides comprising a singlechain immunoglobulin with an immunoglobulin heavy chain and animmunoglobulin light chain. In other embodiments, the plurality ofpolynucleotides comprises a DNA library. In some embodiments, the DNAlibrary is generated from cells of a naïve animal. In some embodiments,the DNA library is generated from cells of an immunized animal.

In some embodiments, the antibody made by the cell library is arecombinant antibody, a monoclonal antibody, a chimeric antibody, ahumanized antibody, a human antibody, or an antibody fragment. In someembodiments, the antibody made by the cell library is an IgA, IgD, IgE,IgG or IgM antibody or a subtype thereof.

In some embodiments, the detection molecule is a protein or fragmentthereof. In some embodiments, the detection molecule is an antigen ofinterest. In some embodiments, the detection molecule is labeled. Incertain embodiments, the cells bound by the detection molecule areidentified by flow cytometry. In some embodiments, the cells bound bythe detection molecule are isolated by FACS.

In one aspect the present invention provides a cell library, each cellcomprising: (a) a first polypeptide comprising any of the membrane-boundpolypeptides described herein, and (b) a second polypeptide comprisingan immunoglobulin heavy chain. In some embodiments, the two polypeptidesare able to form a heterodimeric molecule. In some embodiments, theheterodimeric molecule is expressed on the surface of the cell. In someembodiments, each cell further comprises an immunoglobulin light chain.In some embodiments, the second polypeptide comprises a single chainimmunoglobulin with an immunoglobulin heavy chain and an immunoglobulinlight chain. In some embodiments, the heterodimeric molecule comprises asingle antigen-binding site.

In some embodiments, the present invention provides a cell library, eachcell comprising: (a) a first polypeptide comprising any of themembrane-bound polypeptides described herein, and (b) a secondpolypeptide comprising an immunoglobulin heavy chain constant regioncomprising CH2 and CH3 domains. In some embodiments, the twopolypeptides are able to form a heterodimeric molecule. In someembodiments, the heterodimeric molecule is expressed on the surface ofthe cell. In some embodiments, the second polypeptide comprises animmunoglobulin Fc region. In some embodiments, the second polypeptidecomprises: (a) an immunoglobulin Fc region, and (b) a randomizedpolypeptide. In some embodiments, the second polypeptide comprises: (a)a region that is able to form disulfide bonds, and (b) a randomizedpolypeptide. In some embodiments, the second polypeptide comprises: (a)a region that is able to form disulfide bonds, and (b) a mutagenizedpolypeptide.

In another aspect, the present invention provides methods for screeningany of the cell libraries described herein. In some embodiments, themethod of screening a cell library comprises contacting the cell librarywith a detection molecule (e.g., a target of interest). In someembodiments, the method comprises identifying the cells that are boundby the detection molecule. In some embodiments, the method comprisesisolating the cells that are bound by the detection molecule. In someembodiments, the detection molecule is a protein or fragment thereof. Insome embodiments, the detection molecule is an antigen of interest. Insome embodiments, the detection molecule is a small molecule compound.In certain embodiments, the detection molecule is labeled. In someembodiments, the cells are identified by flow cytometry. In certainembodiments, the cells are isolated by FACS.

In another aspect, the present invention provides methods of screeningfor antibodies. In some embodiments, the method of screening for aspecific antibody comprises contacting the cells or cell librariesdescribed herein with a detection molecule (e.g., a target of interest).In some embodiments, the method of screening for a specific antibodycomprises identifying the cells that are bound by the detectionmolecule. In some embodiments, the method for screening for a specificantibody comprises isolating the cells that are bound by the detectionmolecule. In some embodiments, the method for screening for a specificantibody comprises isolating the antibody from the cells identified bythe detection molecule. In some embodiments, the detection molecule is aprotein or fragment thereof. In some embodiments, the detection moleculeis an antigen of interest. In some embodiments, the detection moleculeis a small molecule compound. In certain embodiments, the detectionmolecule is labeled. In some embodiments, the cells are identified byflow cytometry. In certain embodiments, the cells are isolated by FACS.

In another aspect, the present invention provides antibodies produced,identified, and/or isolated by any of the methods described herein.

BRIEF DESCRIPTIONS OF THE DRAWINGS

FIG. 1A. Schematic of the Membrane-MAb technique compared with otherhybridoma strategies. FIG. 1A depicts a typical hybridoma indicatingthat monoclonal antibody is secreted from the hybridoma.

FIG. 1B depicts a hybridoma that expresses an antibody-binding proteinor “capture protein” (e.g., an Fc receptor or protein A) on its cellsurface. The schematic indicates that monoclonal antibody is secretedfrom the hybridoma. However, the hybridoma has an antibody-bindingprotein on the cell surface that can bind antibody, i.e., antibodysecreted by the cell but also antibody produced by other cells.

FIG. 1C depicts one non-limiting embodiment of the Membrane-MAbstrategy. The hybridoma expresses a heterodimeric molecule thatcomprises a membrane-bound polypeptide covalently associated with animmunoglobulin heavy chain-light chain pair. The immunoglobulin heavychain-light chain pair forms a single antigen-binding siterepresentative of the monoclonal antibody produced by the cell.

FIG. 2A. Generation of a cell expressing a Membrane-MAb polypeptide foruse in fusions. The murine hybridoma fusion partner cell line SP2/0-Ag14was stably transfected with the Membrane-MAb(mIgG1) construct. Theresulting cell line was designated SP2/0-MT. FIG. 2A shows flowcytometry results for cell surface expression of the Membrane-MAb(mIgG1)construct on the SP2/0-MT cells using an isotype negative controlantibody (left panel) and an anti-FLAG antibody (right panel). TheMembrane-MAb(mIgG1) construct has a FLAG tag allowing the polypeptide tobe detected by the anti-FLAG antibody.

FIG. 2B shows flow cytometry results for cell surface expression of theMembrane-MAb(mIgG1) construct on five subclones of the SP2/0-MT cellline.

FIG. 3. Use of the Membrane-MAb technique to identify hybridomasproducing an antibody that binds multiple targets. Shown is a flowcytometry plot of a Membrane-MAb hybridoma library prepared by fusion ofSP-2/0 cells with cells isolated from mice immunized with murine FZD5and FZD8. The cells were incubated with labeled FZD5 and FZD8 proteinsand analyzed. Recombinant FZD5 protein was labeled with Alexa Fluor 488and recombinant FZD8 protein was labeled with Alexa Fluor 647. Hybridomacells that display binding to both FZD5 and FZD8 were identified. (Inthe boxed area labeled “FZD5/8 DP”; DP=double positive).

FIG. 4. Use of the Membrane-MAb technique to identify cells producingantibodies to DDR2. Shown is a flow cytometry plot of a Membrane-MAbhybridoma library prepared by transfection of the Membrane-MAb(mIgG1)construct into an existing DDR2 hybridoma library The cells wereincubated with labeled DDR2 protein and an anti-FLAG antibody andanalyzed. Recombinant DDR2 protein was labeled with Alexa Fluor 488 andthe anti-FLAG antibody was labeled with phycoerythrin (PE). In the boxedarea labeled “DDR2 Pos” are hybridoma cells that display binding to DDR2and the anti-FLAG antibody.

FIG. 5A. Flow cytometry analysis of 293-hMT stable clones. HEK-293 cellswere stably transfected with the Membrane-MAb(hIgG2)-GFP construct. 14clones were screened for GFP expression. FIG. 5 a depicts a flowcytometry analysis for clones 4-9.

FIG. 5B. Flow cytometry analysis of 293-hMT stable clones. HEK-293 cellswere stably transfected with the Membrane-MAb(hIgG2)-GFP construct. 14clones were screened for GFP expression. FIG. 5 b depicts a flowcytometry analysis for clones 10-14.

FIG. 6A. Schematic of cell-based antibody display using Membrane-MAbconstruct. FIG. 6A depicts the Membrane-MAb(hIgG2) andMembrane-MAb(hIgG2)-GFP constructs.

FIG. 6B depicts a single chain antibody vector referred to herein asMAbLib construct. To facilitate generation of antibody libraries, theheavy chain variable region and the light chain variable region areflanked by unique restriction sites.

FIG. 6C depicts the Membrane-MAb(hIgG2)-GFP molecule expressed on thesurface of in HEK-293 cells.

FIG. 6D depicts a non-limiting embodiment of a heterodimeric molecule onthe surface of HEK-293 cells. The heterodimeric molecule is aMembrane-MAb(hIgG2)-GFP protein associated with a single chain antibodymolecule.

FIG. 7. Analysis of expression of heterodimeric antibody molecules onthe surface of HEK-293 cells. Various concentrations of anti-DLL4antibody sc21M18 plasmid DNA were used to transfect HEK-293 cells incombination with the Membrane-MAb(hIgG2)-GFP construct (-X-). Controlswere non-transfected cells (-♦-), cells transfected with sc21M18 DNAonly (-▴-), and cells transfected with Membrane-MAb(hIgG2)-GFP DNA only(-▪-). Cells were incubated for 48 hours, harvested, and screened withan antibody specific antigen (hDLL4-Fc) by FACS. Results are shown asmean fluorescence intensities (MFI).

FIG. 8. Analysis of the use of carrier plasmid to modulate display ofthe antibody molecule. Anti-DLL4 antibody plasmid DNA was mixed with anexcess of an irrelevant antibody plasmid DNA (sc18R5) at various ratiosand used to transfect HEK-293 cells. Cells were incubated for 48 hours,harvested, and screened by FACS. For screening of anti-DLL4 antibody onthe surface, hDLL4-rFc was used as antigen (-♦-) and hJag-rFc proteinwas used as a control (-▪-). Bound antigen was detected with aPE-labeled anti-rabbit Fc antibody and was also used alone as a control(-▴-). The percentage of cells expressing anti-DLL4 antibody on thesurface was determined by FACS.

FIG. 9A. Selection and enrichment of cells expressing anti-DLL4antibody. HEK-293 cells were transfected with a mixture of anti-DLL4antibody plasmid DNA and irrelevant antibody plasmid DNA at a ratio of1:100,000 (sc21M18:sc18R5). Cells were subjected to 4 rounds of sortingand at each round the percentage of cells expressing anti-DLL4 antibodyon the surface was determined by FACS. FIG. 9 a depicts rounds 1-3.

FIG. 9B. Selection and enrichment of cells expressing anti-DLL4antibody. HEK-293 cells were transfected with a mixture of anti-DLL4antibody plasmid DNA and irrelevant antibody plasmid DNA at a ratio of1:100,000 (sc21M18:sc18R5). Cells were subjected to 4 rounds of sortingand at each round the percentage of cells expressing anti-DLL4 antibodyon the surface was determined by FACS. FIG. 9 b depicts rounds 4a and4b.

DETAILED DESCRIPTION

The present invention provides novel membrane-bound polypeptides,including polypeptides that are able to form heterodimeric molecules onthe surface of cells. Related polypeptides and polynucleotides, as wellas cells and cell libraries comprising the polypeptides andpolynucleotides are also provided. Methods of producing host cellscomprising the polypeptides and using these cells to identify andisolate specific individual cells are further provided. Antibodiesproduced by the cells, or produced, isolated, and/or identified by themethods are also provided.

Constructs comprising nucleotides encoding for novel membrane-boundpolypeptides, including Membrane-Mab(mIgG1), Membrane-Mab(hIgG2), andMembrane-Mab(hIgG2)-GFP were generated (Example 1). Cells that expressthe novel membrane-bound polypeptides were produced. The cell lineSP2/0-MT was generated by transfecting the Membrane-Mab(mIgG1) constructinto SP2/0-Ag14 cells, a murine fusion partner cell line (Example 2).The cell line 293-hMT was generated by transfecting theMembrane-Mab(hIgG2)-GFP construct into HEK-293 cells, a human embryonickidney-derived cell line (Example 5). SP2/0-MT cells were used in afusion with cells from an immunized animal to produce a hybridomalibrary expressing heterodimeric antibody molecules on the cell surface.These membrane-bound antibody molecules were used to detect and isolatecells producing antibodies that specifically bound to the targetantigens (Example 3). A polynucleotide encoding the novel polypeptideMembrane-Mab(mIgG1) was transfected into an established hybridomalibrary and the resulting cells were shown to express a heterodimericantibody molecule on their surface. The membrane-bound antibodymolecules were used to detect and isolate cells producing antibodiesthat specifically bound to the target antigen (Example 4). Using both ahybridoma fusion method and a transfection method, the Membrane-MAbtechnique resulted in a dramatic increase in the percentage ofantigen-specific positive clones isolated (91% and 84% positive) ascompared to randomly selected clones (0.6% and 8% positive). TheMembrane-Mab(hIgG2)-GFP construct was transfected into HEK-293 cellswith DNA encoding an anti-DLL4 antibody. Analysis by flow cytometrydemonstrated the presence of a heterodimeric molecule on the surface ofthe transfected cells which bound DLL4 antigen (Example 7). A validationstudy using the Membrane MAb technique demonstrated that cellsexpressing a heterodimeric molecule comprising an anti-DLL4 antibodycould be selected out of a population of cells wherein a differentantibody was expressed in lame excess (Example 9).

I. Definitions

To facilitate an understanding of the present invention, a number ofterms and phrases are defined below.

The term “antibody” as used herein refers to an immunoglobulin moleculethat recognizes and specifically binds to a target, such as a protein,polypeptide, peptide, carbohydrate, polynucleotide, lipid, orcombinations of the foregoing through at least one antigen recognitionsite or antigen-binding site within the variable region of theimmunoglobulin molecule. As used herein, the term “antibody” encompassesintact polyclonal antibodies, intact monoclonal antibodies, antibodyfragments (such as Fab, Fab′, F(ab′)2, and Fv fragments), single chainFv (scFv) mutants, single chain immunoglobulin molecules which includean immunoglobulin heavy chain and an immunoglobulin light chain in theirentirety, multispecific antibodies such as bispecific antibodiesgenerated from at least two intact antibodies, chimeric antibodies,humanized antibodies, human antibodies, fusion proteins comprising anantigen recognition site of an antibody, and any other modifiedimmunoglobulin molecule comprising an antigen recognition site so longas the antibody exhibits the desired biological activity. In addition,the term “antibody” includes monovalent antibody molecules that haveonly one binding site. An antibody can be any of the five major classesof immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses thereof(e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2), based on the identity oftheir heavy chain constant domains referred to as alpha, delta, epsilon,gamma, and mu, respectively.

The term “Fc region” as used herein refers to a C-terminal region of animmunoglobulin heavy chain. The “Fc region” may be a native sequence Fcregion or a variant Fc region. Although the boundaries of the Fc regionof an immunoglobulin heavy chain may vary, the Fc region of animmunoglobulin generally comprises two constant domains, CH2 and CH3,and at least a portion of the hinge region.

The term “antibody fragment” refers to a portion of an intact antibodyand refers to the antigenic determining variable regions of an intactantibody. Examples of antibody fragments include, but are not limitedto, Fab, Fab′, F(ab′)2, and Fv fragments, linear antibodies, scFvantibodies, and multispecific antibodies formed from antibody fragments.

The term “variable region” of an antibody refers to the variable regionof the antibody light chain or the variable region of the antibody heavychain, either alone or in combination. The variable regions of the heavyand light chains each consist of four framework regions connected bythree complementarity determining regions (CDRs), also known as“hypervariable regions”. The CDRs in each chain are held together inclose proximity by the framework regions and, with the CDRs from theother chain, contribute to the formation of the antigen-binding site ofthe antibody. There are at least two techniques for determining CDRs:(1) an approach based on cross-species sequence variability (i.e., Kabatet al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed.,National Institutes of Health, Bethesda Md.); and (2) an approach basedon crystallographic studies of antigen-antibody complexes (Al-Lazikaniet al., 1997, J. Molec. Biol. 273:927-948). In addition, combinations ofthese two approaches can be used in the art to determine CDRs.

The term “monoclonal antibody” refers to a homogeneous antibodypopulation involved in the highly specific recognition and binding of asingle antigenic determinant or epitope. This is in contrast topolyclonal antibodies that typically include a mixture of differentantibodies directed against different antigenic determinants. The term“monoclonal antibody” encompasses both intact and full-length monoclonalantibodies as well as antibody fragments (such as Fab, Fab′, F(ab′)2, Fvfragments), scFv variants, fusion proteins comprising an antibodyportion, and any other modified immunoglobulin molecule comprising anantigen recognition site. “Monoclonal antibody” refers to suchantibodies made by any number of techniques including, but not limitedto, hybridoma production, phage selection, recombinant expression, andtransgenic animals.

The term “humanized antibody” refers to forms of non-human (e.g.,murine) antibodies that are specific immunoglobulin chains, chimericimmunoglobulins, or fragments thereof that contain minimal non-human(e.g., murine) sequences.

The term “human antibody” refers to an antibody produced by a human oran antibody having an amino acid sequence corresponding to an antibodyproduced by a human made using any technique known in the art. Thisdefinition of a human antibody includes intact or full-lengthantibodies, fragments thereof, and/or antibodies comprising at least onehuman heavy and/or light chain polypeptide such as, for example, anantibody comprising a murine light chain polypeptide and a human heavychain polypeptide.

The term “chimeric antibodies” refers to antibodies wherein the aminoacid sequence of the immunoglobulin molecule is derived from two or morespecies. Typically, the variable region of both light and heavy chainscorresponds to the variable region of antibodies derived from onespecies of mammal (e.g., mouse, rat, rabbit) with the desiredspecificity, affinity, and/or capability while the constant regions arehomologous to the sequences in antibodies derived from another species(usually human) to avoid eliciting an immune response in that species.

The terms “epitope” and “antigenic determinant” are used interchangeablyherein and refer to that portion of an antigen capable of beingrecognized and specifically bound by a particular antibody. When theantigen is a polypeptide, epitopes can be formed both from contiguousamino acids (often referred to as “linear epitopes”) and noncontiguousamino acids juxtaposed by tertiary folding of a protein (often referredto as “conformation epitopes”). Epitopes formed from contiguous aminoacids are typically retained upon protein denaturing, whereas epitopesformed by tertiary folding are typically lost upon protein denaturing.An epitope typically includes at least 3, and more usually, at least 5or 8-10 amino acids in a unique spatial conformation.

The term “non-immunoglobulin” as used herein refers to polypeptides thatare not an antibody immunoglobulin chain. As used herein“non-immunoglobulin” encompasses other members of the immunoglobulinsuperfamily.

The terms “specifically binds” and “specific binding” mean that abinding agent or an antibody reacts or associates more frequently, morerapidly, with greater duration, with greater affinity, or with somecombination of the above to an epitope or protein than with alternativesubstances, including unrelated proteins. In certain embodiments,“specifically binds” means, for instance, that an antibody binds to aprotein with a K_(D) of about 0.1 mM or less, but more usually less thanabout 1 μM. In certain embodiments, “specifically binds” means that anantibody binds to a protein with a K_(D) of at least about 0.1 μM orless, at least about 0.01 μM or less, or at least about 1 nM or less.Because of the sequence identity between homologous proteins indifferent species, specific binding can include an antibody thatrecognizes a particular protein in more than one species (e.g., a mouseFZD and a human FZD). Likewise, because of homology between differentproteins in certain regions of the polypeptide sequences, specificbinding can include an antibody (or other polypeptide or agent) thatrecognizes more than one protein (e.g., human FZD5 and human FZD8). Itis understood that an antibody or binding moiety that specifically bindsto a first target may or may not specifically bind to a second target.As such, “specific binding” does not necessarily require (although itcan include) exclusive binding, i.e. binding to a single target. Thus,an antibody may, in certain embodiments, specifically bind to more thanone target. In certain embodiments, the multiple targets may be bound bythe same antigen-binding site on the antibody. For example, an antibodymay, in certain instances, comprise two identical antigen-binding sites,each of which specifically binds the same epitope on two or moreproteins. Generally, but not necessarily, reference to binding meansspecific binding.

The terms “polypeptide” and “peptide” and “protein” are usedinterchangeably herein and refer to polymers of amino acids of anylength. The polymer may be linear or branched, it may comprise modifiedamino acids, and it may be interrupted by non-amino acids. The termsalso encompass an amino acid polymer that has been modified naturally orby intervention; for example, disulfide bond formation, glycosylation,lipidation, acetylation, phosphorylation, or any other manipulation ormodification, such as conjugation with a labeling component. Alsoincluded within the definition are, for example, polypeptides containingone or more analogs of an amino acid (including, for example, unnaturalamino acids), as well as other modifications known in the art. It isunderstood that, because at least some of the polypeptides of thisinvention are based upon antibodies, in certain embodiments, thepolypeptides can occur as single chains or associated chains.

The terms “polynucleotide” and “nucleic acid,” are used interchangeablyherein and refer to polymers of nucleotides of any length, and includeDNA and RNA. The nucleotides can be deoxyribonucleotides,ribonucleotides, modified nucleotides or bases, and/or their analogs, orany substrate that can be incorporated into a polymer by DNA or RNApolymerase. A polynucleotide may comprise modified nucleotides, such asmethylated nucleotides and their analogs. If present, modification tothe nucleotide structure may be imparted before or after assembly of thepolynucleotide. The sequence of nucleotides may be interrupted bynon-nucleotide components. A polynucleotide may be further modifiedafter assembly, such as by conjugation with a labeling component.

“Conditions of high stringency” may be identified by those that: (1)employ low ionic strength and high temperature for washing, for example,0.015M sodium chloride/0.0015M sodium citrate/0.1% sodium dodecylsulfate at 50° C.; (2) employ during hybridization a denaturing agent,such as formamide, for example, 50% (v/v) formamide with 0.1% bovineserum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodiumphosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodiumcitrate at 42° C.; or (3) employ 50% formamide, 5×SSC (0.75M NaCl,0.075M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodiumpyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA (50μg/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., with washes at 42°C. in 0.2×SSC (sodium chloride/sodium citrate) and 50% formamide at 55°C., followed by a high-stringency wash consisting of 0.1×SSC containingEDTA at 55° C.

The terms “identical” or “percent identity” in the context of two ormore nucleic acids or polypeptides, refer to two or more sequences orsubsequences that are the same or have a specified percentage ofnucleotides or amino acid residues that are the same, when compared andaligned (introducing gaps, if necessary) for maximum correspondence, notconsidering any conservative amino acid substitutions as part of thesequence identity. The “percent identity” may be measured using sequencecomparison software or algorithms or by visual inspection. Variousalgorithms and software that may be used to obtain alignments of aminoacid or nucleotide sequences are well-known to those of skill in theart. These include, but are not limited to, BLAST, ALIGN, Megalign,BestFit, and GCG Program. In some embodiments, two nucleic acids orpolypeptides of the invention are substantially identical, meaning theyhave at least 70%, at least 75%, at least 80%, at least 85%, at least90%, and in some embodiments at least 95%, 96%, 97%, 98%, 99% nucleotideor amino acid residue identity, when compared and aligned for maximumcorrespondence, as measured using a sequence comparison algorithm or byvisual inspection. In some embodiments, identity exists over a region ofthe sequences that is at least about 10, at least about 20, at leastabout 40-60, at least about 60-80 residues in length or any integralvalue therebetween. In some embodiments, identity exists over a longerregion than 60-80 residues, such as at least about 90-100 residues, andin some embodiments the sequences are substantially identical over thefull length of the sequences being compared, such as the coding regionof a nucleotide sequence.

A “conservative amino acid substitution” is one in which one amino acidresidue is replaced with another amino acid residue having a similarside chain. Families of amino acid residues having similar side chainshave been defined in the art, including basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). For example, substitution of aphenylalanine for a tyrosine is considered a conservative substitution.Preferably, conservative substitutions in the sequences of thepolypeptides and antibodies of the invention do not abrogate the bindingof the polypeptide or antibody containing the amino acid sequence, tothe antigen(s), i.e., the one or more proteins to which the polypeptideor antibody binds. Methods of identifying nucleotide and amino acidconservative substitutions that do not eliminate antigen binding arewell-known in the art.

The term “vector” refers to a construct that is capable of delivering,and preferably expressing, one or more gene(s) or sequence(s) ofinterest in a host cell. Examples of vectors include, but are notlimited to, viral vectors, naked DNA or RNA expression vectors, plasmid,cosmid or phage vectors, DNA or RNA expression vectors associated withcationic condensing agents, and DNA or RNA expression vectorsencapsulated in liposomes.

As used herein the term “transfection” is used to refer to the uptake offoreign DNA by a cell. A cell has been “transfected” when exogenous DNAhas been introduced inside the cell membrane.

A polypeptide, antibody, polynucleotide, vector, cell, or compositionthat is “isolated” is a polypeptide, antibody, polynucleotide, vector,cell, or composition that is in a form not found in nature. Isolatedpolypeptides, antibodies, polynucleotides, vectors, cells orcompositions include those that have been purified to a degree that theyare no longer in a form in which they are found in nature. In someembodiments, an antibody, polynucleotide, vector, cell, or compositionthat is isolated is substantially pure.

As used herein, “substantially pure” refers to material that is at least50% pure (i.e., free from contaminants), more preferably at least 90%pure, more preferably at least 95% pure, more preferably at least 98%pure, more preferably at least 99% pure.

As used herein, “animal” refers to any animal (e.g., a mammal)including, but not limited to, mice, rats, hamsters, other rodents,rabbits, goats, canines, felines, non-human primates, humans, and thelike.

The term “and/or” as used in a phrase such as “A and/or B” herein isintended to include both A and B; A or B; A (alone) and B (alone).

As used in the present disclosure and claims, the singular forms “a”“an” and “the” include plural forms unless the context clearly dictatesotherwise.

It is understood that wherever embodiments are described herein with thelanguage “comprising” otherwise analogous embodiments described in termsof “consisting of” and/or “consisting essentially of” are also provided.

II. Polypeptides and Polynucleotides

The present invention provides novel polypeptides that aremembrane-bound and which comprise an extracellular portion expressed onthe surface of cells. In some embodiments, the polypeptide comprises:(a) an extracellular portion comprising a dimerization domain, and (b) atransmembrane portion. As used herein, a dimerization domain is anydomain that can facilitate interaction between two polypeptides.Suitable dimerization domains include those of proteins havingamphipathic alpha helices in which hydrophobic residues are regularlyspaced and allow the formation of a dimer by interaction of thehydrophobic residues of each protein. Suitable dimerization domainsinclude those of proteins having cysteine residues that allow theformation of a dimer through formation of disulfide bonds. Dimerizationdomains may include, but are not limited to, immunoglobulin constantregions, leucine zippers, isoleucine zippers, and GCN4 zippers. In someembodiments, the polypeptide comprises: (a) an extracellular portioncomprising an immunoglobulin heavy chain constant region, and (b) atransmembrane portion. In some embodiments, the polypeptide comprises:(a) an extracellular portion comprising a dimerization domain, and (b) aGPI-anchored portion. In some embodiments, the polypeptide comprises:(a) an extracellular portion comprising an immunoglobulin heavy chainconstant region, and (b) a GPI-anchored portion. In some embodiments,the immunoglobulin heavy chain constant region comprises at least oneconstant domain selected from CH2, CH3, and/or CH4. In some embodiments,the immunoglobulin heavy chain constant region comprises at least aportion of a hinge region. In some embodiments, the immunoglobulin heavychain constant region comprises CH2 and CH3 domains. In someembodiments, the immunoglobulin heavy chain constant region comprises ahinge region, CH2 and CH3. In some embodiments, the immunoglobulin heavychain constant region comprises a Fc region. In some embodiments, theimmunoglobulin heavy chain constant region is obtained from an IgA, IgD,IgE, IgG, IgM antibody or a subtype thereof. In some embodiments, theimmunoglobulin heavy chain constant region is obtained from IgG1 orIgG2. In some embodiments, the immunoglobulin heavy chain constantregion is obtained from IgG2. In some embodiments, the immunoglobulinheavy chain constant region is obtained from a human immunoglobulinheavy chain region. In other embodiments, the immunoglobulin heavy chainconstant region is obtained from a mouse immunoglobulin heavy chainconstant region.

In some embodiments, the polypeptide does not comprise an antibodyvariable region. In some embodiments, the polypeptide does not have anantigen-binding site.

The polypeptides are anchored in the cell membrane, at least partially,by a transmembrane portion of the polypeptide or a GPI-membrane anchor.In some embodiments, the transmembrane portion is obtained from a Type Itransmembrane protein. Type I transmembrane proteins are situated sothat their N-terminus is outside of the membrane. Type I transmembraneproteins, may be single pass, meaning they cross the membrane only once,or multi-pass, meaning they cross the membrane sever al times. Thetransmembrane portion may be obtained from a variety of sources,including but not limited to, immunoglobulin and non-immunoglobulinproteins. In some embodiments, the transmembrane portion is taken from aprotein that is part of the immunoglobulin superfamily, including butnot limited to, CD4, CD8, Class I MHC, Class II MHC, CD19, T-cellreceptor a and chains, CD3, zeta chain, ICAM1 (CD54), ICAM2, ICAM3,ICAM4, ICAM5, CD28, CD79a, CD79b, and CD2. In some embodiments, thetransmembrane portion is obtained from a human protein. In someembodiments, the transmembrane portion is obtained from a murineprotein. In some embodiments, the transmembrane portion is obtained froma human CD4.

In some embodiments, the polypeptides further comprise at least aportion of an intracellular domain. In some embodiments, theintracellular domain is obtained from the same protein the transmembraneportion is obtained from. In some embodiments, the intracellular domainis obtained from a different protein than the protein the transmembraneportion is obtained from. In some embodiments, the intracellular domainis modified. In some embodiments, the intracellular domain is modifiedso the normal function of the domain is removed or inactivated.Modifications may include, but are not limited to, removal of a proteinbinding site, removal or inhibition of an activation site, and removalor inhibition of a phosphorylation site. In some embodiments, thepolypeptide comprises a transmembrane and intracellular domain regionfrom human CD4. In other embodiments, the polypeptide comprises atransmembrane and intracellular domain region from human CD4 wherein theintracellular domain has been modified. In some embodiments, theintracellular domain has been modified to remove a lck protein bindingsite.

In some embodiments, the transmembrane portion or transmembrane portionwith intracellular domain is selected from the group consisting of: SEQID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17. Insome embodiments, the transmembrane portion with intracellular domain isSEQ ID NO:15.

In some embodiments, the polypeptides are anchored to the cell membraneby a GPI-membrane anchor. In some embodiments, the polypeptides areanchored to the cell membrane by a GPI linkage where the typicalhydrophobic C-terminal amino acid residues are cleaved from thepropeptide and the C-terminus of the polypeptide is covalently linked toglycosylphosphatidylinositol. The hydrophobic lipid moiety of GPIretains the polypeptide at the cell membrane. In some embodiments, thehydrophobic C-terminal portion of the molecule responsible for GPIlinkage is obtained from CD52, CD55, CD58, CD59, and other similarproteins.

The polypeptides of the present invention are membrane-bound andcomprise an immunoglobulin heavy chain constant region expressed on thesurface of a cell. In some embodiments, the polypeptide is able toassociate with a second polypeptide to form a heterodimeric molecule. Insome embodiments, the second polypeptide comprises an immunoglobulinheavy chain. In some embodiments, the second polypeptide comprises animmunoglobulin heavy chain and an immunoglobulin light chain as a singlechain molecule. In some embodiments, the second polypeptide comprises animmunoglobulin light chain. In some embodiments, the immunoglobulinlight chain is associated with the immunoglobulin heavy chain. In someembodiments, the second polypeptide comprises an immunoglobulin heavychain-light chain pair. As used herein, “an immunoglobulin heavychain-light chain pair” includes a single chain immunoglobulincontaining both an immunoglobulin heavy chain and an immunoglobulinlight chain in one polypeptide. In some embodiments, the immunoglobulinheavy chain-light chain pair comprises a single antigen-binding site. Insome embodiments, the membrane-bound polypeptide associates with animmunoglobulin heavy chain-light chain pair to form a heterodimericantibody molecule, wherein the heterodimeric antibody molecule comprisesone single antigen-binding site. In some embodiments, the heterodimericantibody molecule is a monovalent antibody.

The polypeptides of the present invention may associate with a secondpolypeptide by any number of means including, but not limited to,non-covalent associations or covalent associations. In some embodiments,the polypeptide associates with a second polypeptide by non-covalentbonds, such ionic bonds. In some embodiments, the polypeptide associateswith a second polypeptide by covalent bonds, such as disulfide bonds. Insome embodiments, the polypeptide is able to form at least one disulfidebond with a second polypeptide. In some embodiments, the polypeptideforms one disulfide bond with a second polypeptide. In otherembodiments, the polypeptide forms two disulfide bonds with a secondpolypeptide. In other embodiments, the polypeptide forms three or fourdisulfide bonds with a second polypeptide. In other embodiments, thepolypeptide is able to form at least one disulfide bond with theimmunoglobulin heavy chain constant region of an immunoglobulin heavychain-light chain pair to form a heterodimeric antibody molecule.

In some embodiments, the polypeptides of the present invention are ableto form disulfide bonds with a second polypeptide to form aheterodimeric molecule. By their design, the polypeptides generally arenot able to form disulfide bonds with a polypeptide that is already partof a heterodimeric or a homodimeric molecule. The polypeptides generallyare not able to form disulfide bonds with a secreted antibody molecule.In some embodiments, the polypeptides do not bind secreted antibody. Insome embodiments, the heterodimeric molecule comprising a polypeptide ofthe present invention does not bind antibody. In certain embodiments,the heterodimeric molecule comprising a polypeptide of the presentinvention does not bind secreted antibody.

In some embodiments, the invention provides a polypeptide comprising:(a) an immunoglobulin heavy chain constant region comprising CH2 andCH3; and (b) a transmembrane portion. In certain embodiments, thepolypeptide comprises: (a) an human IgG2 heavy chain constant regioncomprising CH2 and CH3; and (b) a human CD4 transmembrane portion. Incertain embodiments, the polypeptide comprises: (a) an immunoglobulinheavy chain constant region comprising cH2 and CH3; (b) a transmembraneportion; and (c) a fluorescent molecule. In certain embodiments, thepolypeptide comprises: (a) an immunoglobulin heavy chain constant regioncomprising CH2 and CH3; (b) a CD4 transmembrane portion; and (c) afluorescent molecule. In certain embodiments, the polypeptide comprises:(a) an human IgG2 heavy chain constant region comprising CH2 and CH3;(b) a human CD4 transmembrane portion: and (c) a fluorescent molecule.In certain embodiments, the fluorescent molecule is GFP.

In some embodiments, the invention provides a polypeptide comprising:(a) an immunoglobulin heavy chain constant region comprising SEQ IDNO:2, SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:8; and (b) a transmembraneportion comprising SEQ ID NO:13 or SEQ ID NO:16. In some embodiments,the polypeptide comprises an immunoglobulin heavy chain constant regioncomprising SEQ ID NO:2 and a transmembrane portion comprising SEQ IDNO:16. In some embodiments, the polypeptide comprises an immunoglobulinheavy chain constant region comprising SEQ ID NO:2 and a transmembraneportion of SEQ ID NO:17. In some embodiments, the polypeptide comprisesan immunoglobulin heavy chain constant region comprising SEQ ID NO:2 anda transmembrane portion comprising SEQ ID NO:13. In some embodiments,the polypeptide comprises an immunoglobulin heavy chain constant regioncomprising SEQ ID NO:2 and a transmembrane portion of SEQ ID NO:15. Insome embodiments, the polypeptide comprises an immunoglobulin heavychain constant region comprising SEQ ID NO:4 and a transmembrane portioncomprising SEQ ID NO:16. In some embodiments, the polypeptide comprisesan immunoglobulin heavy chain constant region comprising SEQ ID NO:4 anda transmembrane portion of SEQ ID NO:17. In some embodiments, thepolypeptide comprises an immunoglobulin heavy chain constant regioncomprising SEQ ID NO:4 and a transmembrane portion comprising SEQ IDNO:13. In some embodiments, the polypeptide comprises an immunoglobulinheavy chain constant region comprising SEQ ID NO:4 and a transmembraneportion of SEQ ID NO:15. In some embodiments, the polypeptide comprisesan immunoglobulin heavy chain constant region comprising SEQ ID NO:6 anda transmembrane portion comprising SEQ ID NO:16. In some embodiments,the polypeptide comprises an immunoglobulin heavy chain constant regioncomprising SEQ ID NO:6 and a transmembrane portion of SEQ ID NO:17. Insome embodiments, the polypeptide comprises an immunoglobulin heavychain constant region comprising SEQ ID NO:6 and a transmembrane portioncomprising SEQ ID NO:13. In some embodiments, the polypeptide comprisesan immunoglobulin heavy chain constant region comprising SEQ ID NO:6 anda transmembrane portion of SEQ ID NO:15. In some embodiments, thepolypeptide comprises an immunoglobulin heavy chain constant regioncomprising SEQ ID NO:8 and a transmembrane portion comprising SEQ IDNO:16. In some embodiments, the polypeptide comprises an immunoglobulinheavy chain constant region comprising SEQ ID NO:8 and a transmembraneportion of SEQ ID NO:17. In some embodiments, the polypeptide comprisesan immunoglobulin heavy chain constant region comprising SEQ ID NO:8 anda transmembrane portion comprising SEQ ID NO:13. In some embodiments,the polypeptide comprises an immunoglobulin heavy chain constant regioncomprising SEQ ID NO:8 and a transmembrane portion of SEQ ID NO:15.

In some embodiments, the invention provides a polypeptide comprising: anamino acid sequence having at feast about 80% sequence identity to SEQID NO:2, SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:8, and an amino acidsequence having at least 80% sequence identity to SEQ ID NO:13, SEQ IDNO: 14, SEQ ID NO:15, SEQ ID NO:16, or SEQ ID NO:17. In certainembodiments, the polypeptide comprises an amino acid sequence having atleast about 85%, at least about 90%, at least about 95%, at least about97%, or at least about 99% sequence identity to SEQ ID NO:2, SEQ IDNO:4, SEQ ID NO:6, or SEQ ID NO:8, and an amino acid sequence having atleast about 85%, at least about 90%, at least about 95%, at least about97%, or at least about 99% sequence identity to SEQ ID NO:13, SEQ NO:14SEQ ID NO:15, SEQ ID NO:16, or SEQ ID NO:17. In some embodiments, thepolypeptide comprises an amino acid sequence having at least about 95%identity to SEQ ID NO:2 and an amino acid sequence having at least about95% identity to SEQ ID NO:14 or SEQ ID NO:15. In some embodiments, thepolypeptide comprises an amino acid sequence having at least about 95%identity to SEQ ID NO:4 and an amino acid sequence having at least about95% identity to SEQ ID NO:14 or SEQ ID NO:15. In some embodiments, thepolypeptide comprises an amino acid sequence having at least about 95%identity to SEQ ID NO:6 and an amino acid sequence having at least about95% identity to SEQ ID NO:14 or SEQ ID NO:15. In some embodiments, thepolypeptide comprises an amino acid sequence having at least about 95%identity to SEQ ID NO:8 and an amino acid sequence having at least about95% identity to SEQ ID NO:14 or SEQ ID NO:15.

In certain embodiments, the polypeptide comprises an amino acid sequencehaving at least 80% sequence identity to SEQ ID NO:10, SEQ ID NO:12, SEQID NO:28. In certain embodiments, the polypeptide comprises an aminoacid sequence having at least about 85%, at least about 90%, at leastabout 95%, at least about 97%, or at least about 99% sequence identityto SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:28. In some embodiments, theinvention provides a polypeptide comprising SEQ ID NO:10, SEQ ID NO:12,or SEQ ID NO:28.

The polypeptides of the present invention contain a signal sequence thatdirects the transport of the proteins. Signal sequences (also referredto as signal peptides or leader sequences) are located at the N-terminusof nascent polypeptides. They target the polypeptide to the endoplasmicreticulum and the proteins are sorted to their destinations, forexample, to the inner space of an organelle, to an interior membrane, tothe cell's outer membrane, or to the cell exterior via secretion. Mostsignal sequences are cleaved from the protein by a signal peptidaseafter the proteins are transported to the endoplasmic reticulum. Thecleavage of the signal sequence from the polypeptide usually occurs at aspecific site in the amino acid sequence and is dependent upon aminoacid residues within the signal sequence. Although there is usually onespecific cleavage site, more than one cleavage site may be recognizedand/or used by a signal peptidase resulting in a non-homogenousN-terminus of the polypeptide. For example, the use of differentcleavage sites within a signal sequence can result in a polypeptideexpressed with different N-terminal amino acids. Accordingly, in someembodiments, the polypeptides as described herein may comprise a mixtureof polypeptides with different N-termini. In some embodiments, theN-termini differ in length by 1, 2, 3, 4, or 5 amino acids. In someembodiments, the polypeptide is substantially homogeneous, i.e., thepolypeptides have the same N-terminus. In some embodiments, the signalsequence of the polypeptide comprises one or more (e.g., one, two,three, four, five, six, seven, eight, nine, ten, etc.) amino acidsubstitutions and/or deletions. In some embodiments, the signal sequenceof the polypeptide comprises amino acid substitutions and/or deletionsthat allow one cleavage site to be dominant, thereby resulting in asubstantially homogeneous polypeptide with one N-terminus. Variousalgorithms and software that can be used to predict signal peptidasecleavage sites are known in the art and are publicly available (e.g.,SignalP software).

In certain embodiments, the polypeptide comprises an amino acid sequencehaving at least 80% sequence identity to SEQ ID NO:22, SEQ ID NO:23, orSEQ ID NO:26. In certain embodiments, the polypeptide comprises an aminoacid sequence having at least about 85%, at least about 90%, at leastabout 95%, at least about 97%, or at least about 99% sequence identityto SEQ ID NO:22, SEQ ID NO:23, or SEQ ID NO:26. In some embodiments, thepolypeptide (before signal sequence cleavage) comprises SEQ ID NO:22,SEQ ID NO:23, or SEQ ID NO:26.

In certain embodiments, the polypeptide comprises an amino acid sequencehaving at least 80% sequence identity to SEQ ID NO:30, SEQ ID NO:31, orSEQ ID NO:32. In certain embodiments, the polypeptide comprises an aminoacid sequence having at least about 85%, at least about 90%, at leastabout 95%, at least about 97%, or at least about 99% sequence identityto SEQ ID NO:30, SEQ ID NO:31, or SEQ ID NO:32. In some embodiments, thepolypeptide comprises SEQ ID NO:32. In some embodiments, the polypeptideconsists essentially of SEQ ID NO:32. In some embodiments, thepolypeptide comprises SEQ ID NO:30. In some embodiments, the polypeptideconsists essentially of SEQ ID NO:30. In some embodiments, thepolypeptide comprises SEQ ID NO:31. In some embodiments, the polypeptideconsists essentially of SEQ ID NO:31. A polypeptide that “consistsessentially of” certain amino acids or is “consisting essentially of”certain amino acids may, in some embodiments, include one or more (e.g.,one, two, three, four or more) additional amino acids, so long as theadditional amino acids do not materially affect the function of thepolypeptide. A polypeptide that “consists essentially of” certain aminoacids or is “consisting essentially of” certain amino acids may, in someembodiments, be reduced by one or more (e.g., one, two, three, four ormore) amino acids, so long as the missing amino acids do not materiallyaffect the function of the polypeptide.

In some embodiments, the invention provides an antibody moleculecomprising any of the polypeptides described herein. In someembodiments, the antibody molecule further comprises a secondpolypeptide comprising an immunoglobulin heavy chain. In someembodiments, the antibody molecule further comprises an immunoglobulinlight chain. In some embodiments, the antibody molecule farthercomprises a second polypeptide comprising a single chain immunoglobulinwith an immunoglobulin heavy chain and an immunoglobulin light chain. Incertain embodiments, the antibody comprises a single antibody bindingsite (i.e., monovalent antibody).

In some embodiments, the invention provides a heterodimeric moleculecomprising any of the polypeptides described herein. In someembodiments, the heterodimeric molecule further comprises a secondpolypeptide comprising a dimerization domain. In some embodiments, thesecond polypeptide comprises an immunoglobulin constant region. In someembodiments, the second polypeptide comprises an immunoglobulin heavychain. In some embodiments, the second polypeptide comprises animmunoglobulin light chain. In some embodiments, the second polypeptidecomprises a single chain immunoglobulin with an immunoglobulin heavychain and an immunoglobulin light chain. In certain embodiments, theheterodimeric molecule comprises a single antibody binding site (i.e.,monovalent antibody). In certain embodiments, the invention provides aheterodimeric molecule comprising: (a) a first polypeptide comprising anextracellular portion comprising a dimerization domain, and atransmembrane portion, and (b) a second polypeptide comprising adimerization domain. In certain embodiments, the first and seconddimerization domains are the same. In certain embodiments, the first andsecond dimerization domains are different.

In some embodiments, a heterodimeric molecule comprises (a) a firstpolypeptide comprising (i) an extracellular portion comprising a firstdimerization domain; and (ii) a transmembrane portion; and (b) a secondpolypeptide comprising a second dimerization domain. In someembodiments, the heterodimeric molecule comprises a first dimerizationdomain which is an immunoglobulin constant region. In some embodiments,the heterodimeric molecule comprises a first dimerization domain whichis an immunoglobulin heavy chain constant region. In some embodiments,the heterodimeric molecule comprises a second dimerization domain whichis an immunoglobulin constant region. In some embodiments, theheterodimeric molecule comprises a second dimerization domain which isan immunoglobulin heavy chain constant region. In certain embodiments,the heterodimeric molecule comprises a first polypeptide comprising anyof the polypeptides described herein.

The polypeptides described, herein can be produced by any suitablemethod known in the art. Such methods range from direct proteinsynthesis methods to constructing a DNA sequence encoding polypeptidesequences and expressing those sequences in a suitable host. In someembodiments, a DNA sequence is constructed using recombinant technologyby isolating or synthesizing a DNA sequence encoding a wild-type proteinof interest. Optionally, the sequence can be mutagenized bysite-specific mutagenesis to provide functional variants thereof.

In some embodiments, a DNA sequence encoding a polypeptide of interestmay be constructed by chemical synthesis using an oligonucleotidesynthesizer. Oligonucleotides can be designed based on the amino acidsequence of the desired polypeptide and by selecting those codons thatare favored in the host cell in which the recombinant polypeptide ofinterest will be produced. Standard methods can be applied to synthesizea polynucleotide sequence encoding a polypeptide of interest. Forexample, a complete amino acid sequence can be used to construct aback-translated gene. In some embodiments, a DNA oligomer containing anucleotide sequence coding for the particular polypeptide can besynthesized. For example, several small oligonucleotides coding forportions of the desired polypeptide can be synthesized and then ligated.The individual oligonucleotides typically contain 5′ or 3′ overhangs forcomplementary assembly.

Once assembled (by recombinant technology, chemical synthesis, oranother method), the polynucleotide sequences encoding a particularpolypeptide of interest can be inserted into an expression vector andoperatively linked to an expression control sequence appropriate forexpression of the polypeptide in a desired host. Proper assembly can beconfirmed by nucleotide sequencing, restriction mapping, and/orexpression of a biologically active polypeptide in a suitable host. Asis well-known in the art, in order to obtain high expression levels of atransfected gene in a host, the gene must be operatively linked totranscriptional and translational expression control sequences that arefunctional in the chosen expression host.

In certain embodiments, recombinant expression vectors are used toamplify and express DNA encoding the polypeptides described herein. Forexample, recombinant expression vectors can be replicable DNA constructsthat have synthetic or cDNA-derived DNA fragments encoding apolypeptide, operatively linked to suitable transcriptional ortranslational regulatory elements derived from mammalian, microbial,viral or insect genes. A transcriptional unit generally comprises anassembly of (1) a regulatory element or elements having a role in geneexpression, for example, transcriptional promoters and/or enhancers, (2)a structural or coding sequence that is transcribed into mRNA andtranslated into protein, and (3) appropriate transcription andtranslation initiation and termination sequences. Regulatory elementscan include an operator sequence to control transcription. The abilityto replicate in a host, usually conferred by an origin of replication,and a selection gene to facilitate recognition of transformants canadditionally be incorporated. DNA regions are “operatively linked” whenthey are functionally related to each other. For example, DNA for asignal peptide (secretory leader) is operatively linked to DNA for apolypeptide if it is expressed as a precursor that participates in thesecretion of the polypeptide; a promoter is operatively linked to acoding sequence if it controls the transcription of the sequence; or aribosome binding site is operatively linked to a coding sequence if itis positioned so as to permit translation. Structural elements intendedfor use in yeast expression systems include a leader sequence enablingextracellular secretion of translated protein by a host cell.Alternatively, where recombinant protein is expressed without a leaderor transport sequence, it can include an N-terminal methionine residue.This residue can optionally be subsequently cleaved from the expressedrecombinant protein to provide a final product.

The choice of an expression vector and control elements depends upon thechoice of host. A wide variety of expression host/vector combinationscan be employed. Useful expression vectors for eukaryotic hosts include,for example, vectors comprising expression control sequences from SV40,bovine papilloma virus, adenovirus and cytomegalovirus. Usefulexpression vectors for bacterial hosts include known bacterial plasmids,such as plasmids from E. coli, including pCR1, pBR322, pMB9 and theirderivatives and wider host range plasmids, such as M13 and otherfilamentous single-stranded DNA phages.

Some expression vectors for eukaryotic hosts may have the ability tointegrate into the host genome and some expression vector for eukaryotichosts may persist in the nucleus as extrachromosomal entities. In someembodiments, episomal vectors may offer advantages, such as transfectionof multiple copies per cell resulting in high expression of thepolynucleotide of interest and/or higher transfection efficiency.

Suitable host cells for expression of the polypeptides as describedherein include prokaryotes, yeast, insect or higher eukaryotic cellsunder the control of appropriate promoters. Prokaryotes includegram-negative or gram-positive organisms, for example, E. coli orBacilli. Higher eukaryotic cells include established cell lines ofmammalian origin as described below. Cell-free translation systems canalso be employed.

Various mammalian or insect cell culture systems are used to expressrecombinant polypeptides. In some embodiments, expression of recombinantproteins in mammalian cells is preferred because such proteins aregenerally correctly folded, appropriately modified and completelyfunctional. Examples of suitable mammalian host cell lines include, butare not limited to, COS-7 (monkey kidney-derived), L-929 (murinefibroblast-derived), C127 (murine mammary tumor-derived), 3T3 (murinefibroblast-derived), CHO (Chinese hamster ovary-derived), HEK-293 (humanembryonic kidney-derived), HeLa (human cervical cancer-derived) and BHK(hamster kidney fibroblast-derived) cell lines. In some embodiments,variants of a cell line may be used. For example, 293T cells are HEK-293cells that express the SV40 Large T-antigen, which allows for episomalreplication of transfected plasmids containing the SV40 origin ofreplication. Mammalian expression vectors can comprise non-transcribedelements such as an origin of replication, a suitable promoter andenhancer linked to the gene to be expressed, and other 5′ or 3′ flankingnon-transcribed sequences, and 5′ or 3′ non-translated sequences, suchas necessary ribosome binding sites, a polyadenylation site, splicedonor and acceptor sites, and transcriptional termination sequences. Insome embodiments, baculovirus systems are used for production ofheterologous proteins in insect cells. These methods and techniques arewell known to those of skill in the art (see, e.g., Luckow and Summers,1988, Bio/Technology, 6:47).

In certain embodiments, the polypeptides described herein are isolated.In certain embodiments, the polypeptides described herein aresubstantially pure.

The proteins expressed by a host cell can be purified according to anysuitable method. Such methods include chromatography (e.g., ionexchange, affinity and sizing column chromatography), centrifugation,differential solubility, or by any other standard technique for proteinpurification. Affinity tags such as hexa-histidine, maltose bindingdomain, influenza coat sequence and glutathione-S-transferase can beattached to the protein to allow easy purification by passage over anappropriate affinity column. Isolated proteins can also be physicallycharacterized using such techniques as proteolysis, high performanceliquid chromatography (HPLC), nuclear magnetic resonance and x-raycrystallography.

In some embodiments, supernatants from expression systems that secreterecombinant protein into culture media can be first concentrated using acommercially available protein concentration filter, for example, anAmicon or Millipore Pellicon ultrafiltration unit. Following theconcentration step, the concentrate can be applied to a suitablepurification matrix. In some embodiments, an anion exchange resin can beemployed, for example, a matrix or substrate having pendantdiethylaminoethyl (DEAE) groups. The matrices can be acrylamide,agarose, dextran, cellulose or other types commonly employed in proteinpurification. In some embodiments, a cation exchange step can beemployed. Suitable cation exchangers include various insoluble matricescomprising sulfopropyl or carboxymethyl groups. In some embodiments, ahydroxyapatite (CHT) media can be employed, including but not limitedto, ceramic hydroxyapatite. In some embodiments, one or morereversed-phase HPLC steps employing hydrophobic RP-HPLC media, e.g.,silica gel having pendant methyl or other aliphatic groups, can beemployed to further purify a protein. Some or all of the foregoingpurification steps, in various combinations, can be employed to providea homogeneous recombinant protein.

In certain embodiments, the invention provides polynucleotidescomprising polynucleotides that encode a polypeptide comprising animmunoglobulin heavy chain constant region and a transmembrane portion.In certain embodiments, the invention provides polynucleotidescomprising polynucleotides that encode a polypeptide comprising animmunoglobulin heavy chain constant region and a GPI-membrane portion.The phrase “polynucleotides that encode a polypeptide” encompasses apolynucleotide that includes only coding sequences for the polypeptide,as well as a polynucleotide that includes additional coding and/ornon-coding sequences. The polynucleotides of the invention can be in theform of RNA or in the form of DNA. DNA includes cDNA, genomic DNA, andsynthetic DNA; and can be double-stranded or single-stranded, and ifsingle-stranded, can be the coding strand or non-coding (anti-sense)strand.

In some embodiments, a polynucleotide comprises a polynucleotideencoding a polypeptide comprising SEQ ID NO:10. In some embodiments, thepolynucleotide comprises a polynucleotide encoding a polypeptidecomprising SEQ ID NO:12. In some embodiments, the polynucleotidecomprises a polynucleotide encoding a polypeptide comprising SEQ IDNO:28. In some embodiments, the polynucleotide comprises apolynucleotide encoding a polypeptide (with or without a signalsequence) comprising SEQ ID NO:22, SEQ ID NO:23, or SEQ ID NO:26. Insome embodiments, the polynucleotide comprises a polynucleotide encodinga polypeptide comprising SEQ ID NO:30. In some embodiments, thepolynucleotide comprises a polynucleotide encoding a polypeptidecomprising SEQ ID NO:31. In some embodiments, the polynucleotidecomprises a polynucleotide encoding a polypeptide comprising SEQ IDNO:32. In some embodiments, the polynucleotide comprises apolynucleotide encoding a polypeptide of SEQ ID NO:30. In someembodiments, the polynucleotide comprises a polynucleotide encoding apolypeptide of SEQ ID NO:31. In some embodiments, the polynucleotidecomprises a polynucleotide encoding a polypeptide of SEQ ID NO:32.

In some embodiments, a polynucleotide comprises a polynucleotide of SEQID NO:9, SEQ ID NO:11, or SEQ ID NO:29. In some embodiment, apolynucleotide comprises a polynucleotide of SEQ ID NO:24, SEQ ID NO:25,or SEQ ID NO:27. In certain embodiments, a polynucleotide comprises apolynucleotide having a sequence of at least 80% identical, at least 85%identical, at least about 90% identical, at least about 95% identical,and in some embodiments, at least 96%, 97%, 98% or 99% identical to apolynucleotide of SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:24, SEQ ID NO:25,SEQ ID NO:27, or SEQ ID NO:29.

Also provided is a polynucleotide that comprises a polynucleotide thathybridizes to SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:24, SEQ ID NO:25, SEQID NO:27, or SEQ ID NO:29. In some embodiments, the hybridization isunder condition of high stringency.

In certain embodiments, the polynucleotides comprise the coding sequencefor the mature polypeptide fused in the same reading frame to apolynucleotide that aids, for example, in expression and/or secretion ofa polypeptide from a host cell. For example, a leader or signal sequencefunctions as a sequence for controlling transport of a polypeptide tothe cell surface, and secretion from the cell if the polypeptide is asecretory protein. The polypeptide having a leader sequence (or signalsequence) is a preprotein and can have the leader sequence cleaved bythe host cell to produce the mature form of the polypeptide. Thepolynucleotides can also encode for a proprotein that is the matureprotein plus additional 5′ amino acid residues. A mature protein havinga prosequence is a proprotein and is an inactive form of the protein.Once the prosequence is cleaved an active mature protein remains.

In certain embodiments the polynucleotides comprise the coding sequencefor the mature polypeptide fused in the same reading frame to a markeror tag sequence that allows, for example, for purification and/oridentification of the expressed polypeptide. In some embodiments, when abacterial host is used, the marker sequence can be a hexa-histidine tagsupplied by a pQE-9 vector to provide for purification of the maturepolypeptide. In other embodiments, when a mammalian host is used (e.g.,COS-7 cells) the marker sequence can be a hemagglutinin (HA) tag derivedfrom the influenza hemagglutinin protein. In some embodiments, themarker sequence is “FLAG”, a peptide of sequence DYKDDDDK (SEQ ID NO:17)that can also be used in conjunction with other affinity tags.

The present invention further relates to variants of the hereinabovedescribed polynucleotides encoding, for example, fragments, analogs,and/or derivatives of the polypeptides.

In certain embodiments, the present invention provides polynucleotidescomprising polynucleotides having a nucleotide sequence at least 80%identical, at least 85% identical, at least 90% identical, at least 95%identical, and in some embodiments, at least 96%, 97%, 98% or 99%identical to a polynucleotide encoding a polypeptide described herein.

As used herein, the phrase a polynucleotide having a nucleotide sequenceat least, for example, 95% “identical” to a reference nucleotidesequence is intended to mean that the nucleotide sequence of thepolynucleotide is identical to the reference sequence except that thepolynucleotide sequence can include up to five point mutations per each100 nucleotides of the reference nucleotide sequence. In other words, toobtain a polynucleotide having a nucleotide sequence at least 95%identical to a reference nucleotide sequence, up to 5% of thenucleotides in the reference sequence can be deleted or substituted withanother nucleotide, or a number of nucleotides up to 5% of the totalnucleotides in the reference sequence can be inserted into the referencesequence. These mutations of the reference sequence can occur at the 5′or 3′ terminal positions of the reference nucleotide sequence oranywhere between those terminal positions, interspersed eitherindividually among nucleotides in the reference sequence or in one ormore contiguous groups within the reference sequence.

The polynucleotide variants can contain alterations in the codingregions, non-coding regions, or both. In some embodiments, thepolynucleotide variants contain alterations that produce “silent”substitutions, additions, or deletions, but do not alter the propertiesor activities of the encoded polypeptide. In some embodiments,polynucleotide variants contain “silent” substitutions due to thedegeneracy of the genetic code. Polynucleotide variants can be producedfor a variety of reasons, for example, to optimize codon expression fora particular host (e.g., change codons in the human mRNA to thosepreferred by a bacterial host such as E. coli).

In certain embodiments, the polynucleotides described herein areisolated. In certain embodiments, the polynucleotides described hereinare substantially pure.

Vectors and cells comprising the polynucleotides described herein arealso provided. In some embodiments, an expression vector comprises apolynucleotide molecule. In some embodiments, a host cell comprises anexpression vector comprising the polynucleotide molecule. In someembodiments, a host cell comprises a polynucleotide molecule. In someembodiments, a host cell comprises a polypeptide encoded by thepolynucleotide molecule. In some embodiments, a host cell produces apolypeptide encoded by the polynucleotide molecule.

III. Cells

The present invention also provides cells that express the polypeptidesdescribed herein and provides methods of producing the cells. The cells(e.g., host cells) used to make the cells described herein include allmammalian cells, cell lines, and cell cultures. In some embodiments, thecells are derived from mammals, such as mice, rats, or other rodents, orfrom primates, such as humans or monkeys. In some embodiments, the cellis a murine cell. In some embodiments, the cell is a human cell. Inother embodiments, the cell is a mammalian germ cell or a somatic cell.In other embodiments, the cell is a primary cell culture or animmortalized cell line. The mammalian cells are typically grown in cellculture. In some embodiments, the cells are adhered to a solid surface.In other embodiments, the cells are grown in suspension.

A wide variety of host cells may be transfected with polynucleotidesand/or vectors comprising polynucleotides encoding for the polypeptidesof the present invention. In some embodiments, the cells will beimmortalized eukaryotic cells including, but not limited to, SP2/0,SP2/0-Ag14, NS/0, YB2/0, K6H6/B5, NS-1, FO, Y3/Ag 1.2.3, P3X63Ag8.653,other myelomas, hybridomas, Chinese hamster ovary cells (CHO), HeLacells, baby hamster kidney cells (BHK), CV-1 cells, 3T3 cells, L cells,TC7 cells, and human embryonic kidney cells (HEK-293 and 293T). In someembodiments, the cells are known fusion partner cell lines. In someembodiments, the cells are immortalized cell lines that fuseefficiently, support high level expression of antibodies, and aresensitive to a selection medium. These cell lines can include, but arenot limited to, Sp2/0, SP2/0-Ag14, YB2/0, K6H6/B5, NS-1, FO, Y3/Ag1.2.3, and P3X63Ag8.653. In some embodiments, the cells are myelomasthat do not express immunoglobulin chains. In some embodiments, thecells are a hybridoma or hybridoma library. In certain embodiments, thecells are human cells. In certain embodiments, the cells are HEK-293cells or a variant thereof (e.g., 293T cells). In certain embodiments,the cells are CHO cells. In certain embodiments, the cells are murinecells. In some embodiments, the cells are SP2/0 or a variant thereof(e.g., SP110-Ag14).

A cell expressing a polypeptide of the present invention may be made bya variety of methods known to one of skill in the art. Thepolynucleotides, vectors and/or constructs described herein can beintroduced into suitable host cells by a variety of methods. In general,transfection or infection with a vector is used to obtain mammaliancells that express the polypeptides of the invention.

In some embodiments, the polynucleotide is introduced into a cell bytransfection. In some embodiments, the transfection is a transientexpression, usually resulting in expression of the transfectedpolypeptide for a limited time period. In other embodiments, thetransfection is a stable transfection, resulting in permanent expressionof the transfected polypeptide. In some embodiments, stable transfectionis accompanied by integration of the input DNA into the cellular genome.In some embodiments, the stable transfection results in episomalmaintenance and replication of the input DNA.

DNA can be introduced into eukaryotic cells via conventionaltransfection techniques. The term “transfection” refers to a variety oftechniques known to one of skill in the art for introducing foreignpolynucleotides (e.g., DNA) into a host cell. These techniques include,but are not limited to, calcium phosphate or calcium chlorideco-precipitation, DEAE-dextran-mediated transfection, liposome-mediatedtransfection, electroporation, and microinjection. These and othersuitable methods for transfecting host cells can be found in manystandard laboratory manuals. In some embodiments, cells are transfectedusing liposome-mediated transfection or “lipofection”. Lipofection is alipid-based transfection technology wherein the nucleic acids associatewith a lipid-based transfection reagent resulting in tight compactionand protection of the nucleic acids. The main advantages of lipofectionare high efficiency, the ability to transfect all types of nucleic acidsin a wide range of cell types, ease of use, reproducibility, and lowtoxicity. In addition, lipofection is suitable for all transfectionapplications including, but not limited to, transient, stable,co-transfection, sequential, or multiple transfections.

In some embodiments, the cells are stably transfected with apolynucleotide expressing the polypeptides described herein. For stabletransfection of mammalian cells, it is known that, depending upon theexpression vector and transfection technique used, only a small fractionof cells may integrate the DNA into their genome. In transfection ofmammalian cells with an episomal vector, a larger fraction of cells mayincorporate and maintain the DNA. In order to identify and select thesecells, a gene that encodes a selectable marker (e.g., resistance toantibiotics) is generally introduced into the host cells along with thegene(s) of interest. Various selectable markers include those thatconfer resistance to drugs, such as G418, hygromycin and methotrexate.Polynucleotides encoding a selectable marker can be introduced into ahost cell on the same vector as that encoding the polypeptide or can beintroduced on a separate vector. Cells stably transfected with theintroduced polynucleotides can be identified by drug selection (e.g.,cells that have incorporated the selectable marker gene will survive,while the other cells die).

In some embodiments, the polynucleotide is introduced into a cell by avector. Vectors can be derived from viral genomes that yield virions orvirus-like particles that may or may not replicate independently asextrachromosomal elements. Virion particles containing a polynucleotideencoding a desired polypeptide can be introduced into host cells byinfection. In some embodiments, the viral vector becomes integrated intothe cellular genome. Viral vectors for transformation of mammalian cellsinclude, but are not limited to, SV40 vectors, and vectors based onpapillomavirus, adenovirus, Epstein-Barr virus, vaccinia virus, andretroviruses, such as Rous sarcoma virus, or a mouse leukemia virus,such as Moloney murine leukemia virus.

Also provided are methods of producing cells expressing the polypeptidesdescribed herein. In some embodiments, provided herein are methods ofproducing cells, comprising transfecting cells with a polynucleotide orvector encoding a polypeptide described herein. In some embodiments, thetransfected cells express the polypeptide. In some embodiments, thecells are transiently transfected. In some embodiments, the cells arestably transfected. In some embodiments, the polynucleotide encoding apolypeptide is integrated into the genome of the cell. In someembodiments, the transfected polynucleotide encoding a polypeptide isstably expressed in the cell. In other embodiments, the polypeptide isexpressed on the surface of the transfected cells.

In some embodiments, the methods further comprise detecting expressionof the polypeptide. The transfected cells can be assayed for expressionof the polypeptide by any method known in the art. The assays which canbe used include, but are not limited to, competitive and non-competitiveassay systems using techniques such as Biacore analysis, flow cytometry,FACS analysis, immunofluorescence, immunocytochemistry, Western blotanalysis, radioimmunoassay, ELISA, “sandwich” immunoassay,immunoprecipitation assay, precipitation reaction, gel diffusionprecipitin reaction, immunodiffusion assay, agglutination assay,complement-fixation assay, immunoradiometric assay, fluorescentimmunoassay, and protein A immunoassay. Such assays are routine and wellknown in the art (see, e.g., Ausubel et al., eds, 1994, CurrentProtocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., NewYork).

In some embodiments, the methods comprise detecting expression of thepolypeptide on the surface of the cell. In some embodiments, the cellsare contacted with a detection molecule. In some embodiments, thedetection molecule is an antibody to an immunoglobulin heavy chainconstant region. In some embodiments, the detection molecule is labeled.In some embodiments, the detection molecule is labeled with afluorophore or a chromophore. In some embodiments, the methods comprisedetecting expression of the polypeptide using flow cytometry. In someembodiments, the methods comprise identifying the cells that are boundby the detection molecule by flow cytometry. As used herein, the phrase“a detection molecule” includes, but is not limited to, a population ofdetection molecules. For example, a detection molecule may be a solutionof antibodies, or a solution of proteins or protein fragments.Similarly, the phrase “cells bound by a detection molecule” includes,but is not limited to, cells bound by a number of detection molecules.

Flow cytometry and FACS are techniques known to those of skill in theart. Flow cytometry uses the principles of light scattering, lightexcitation and emission of fluorochrome molecules to generate specificmulti-parameter data from cells. The instruments make measurements oncells as the cells flow in a stream one by one through a sensing point.Flow cytometry can be used for purification of cell populations orisolation of individual cells by fluorescence-activated cell sorting orFACS. In some embodiments, the cells are analyzed at 10-20,000 cells persecond. In some embodiments, the cells are analyzed at 1,000-10,000cells per second. In certain embodiments, the cells are analyzed at100-1000 cells per second. In some embodiments, about 1×10⁶ to 1×10⁸cells are analyzed. In some embodiments, about 1×10⁶ cells are analyzed.In certain embodiments, about 5×10⁶ cells are analyzed. In someembodiments, about 1×10⁷ cells are analyzed. In some embodiments, about2.5×10⁷ cells are analyzed. In some embodiments, the cells are analyzedand sorted. For example, about 2.5×10⁷ cells can be run through a FACSinstrument and analyzed. Target cells can be sorted into the wells ofabout eight 96 wells, and 600 to 700 individual clones can be isolated.Thus, the use of flow cytometry allows for rapid screening of largenumbers of cells. Similarly, the use of FACS allows for detection andsorting of a large number of cells and isolation of individual cells ina rapid manner.

In some embodiments, the methods of producing cells comprisetransfecting cells with a polynucleotide or vector encoding apolypeptide described herein. In some embodiments, the transfected cellsexpress the polypeptide. In some embodiments, the method comprisesdetecting expression of the polypeptide on the surface of the cells. Insome embodiments, the method comprises selecting cells that express thepolypeptide. In some embodiments, the cells are contacted with adetection molecule. In some embodiments, the detection molecule is anantibody to an immunoglobulin heavy chain constant region. In someembodiments, the detection molecule is a target of interest (e.g., aprotein or fragment thereof). In some embodiments, the detectionmolecule is labeled. In some embodiments, the detection molecule islabeled with a fluorophore or a chromophore. In some embodiments, themethods comprise identifying the cells that are bound by the detectionmolecule by flow cytometry. In some embodiments, the methods compriseisolating the cells that are bound by the detection molecule by FACS.

Thus, the invention provides cells comprising a polynucleotide encodingfor a polypeptide described herein. In some embodiments, the cells areproduced by any of the methods described herein. In some embodiments,the cells comprise a vector comprising a polynucleotide encoding for apolypeptide described herein. In some embodiments, the cells comprise apolypeptide described herein. In some embodiments, the cells comprise apolypeptide comprising an immunoglobulin heavy chain constant region anda non-immunoglobulin transmembrane portion. In some embodiments, thepolypeptide is membrane-bound and expressed on the surface of the cells.

The present invention also provides cells that produce a heterodimericmolecule. The heterodimeric molecules are membrane-bound, expressed atthe cell surface and are not secreted from the cell. In someembodiments, the heterodimeric molecule comprises a polypeptidedescribed herein and further comprises at least one additionalpolypeptide. In some embodiments, the additional polypeptide comprisesan immunoglobulin heavy chain constant region comprising CH2 and CH3domains. In some embodiments, the additional polypeptide comprises animmunoglobulin Fc region. In some embodiments, the additionalpolypeptide comprises an immunoglobulin heavy chain. In certainembodiments, the at least one additional polypeptide comprises animmunoglobulin heavy chain and an immunoglobulin light chain. In certainembodiments, the at least one additional polypeptide comprises a singlechain immunoglobulin with an immunoglobulin heavy chain and animmunoglobulin light chain. In some embodiments, the additionalpolypeptide is an antibody.

In some embodiments, the heterodimeric molecule is a membrane-boundpolypeptide covalently associated with a second polypeptide, wherein themembrane-bound polypeptide comprises: (a) an immunoglobulin heavy chainconstant region, and (b) a transmembrane portion. In some embodiments,the membrane bound polypeptide associates with a second polypeptide byforming disulfide bonds. In some embodiments, the membrane-boundpolypeptide forms at least one disulfide bond with a second polypeptideto form the heterodimeric molecule.

In some embodiments, the heterodimeric molecule is a membrane-boundpolypeptide covalently associated with an immunoglobulin heavy chain,wherein the membrane-bound polypeptide comprises: (a) an immunoglobulinheavy chain constant region, and (b) a transmembrane portion. In someembodiments, the membrane-bound polypeptide associates with theimmunoglobulin heavy chain by forming disulfide bonds. In someembodiments, the membrane-bound polypeptide forms at least one disulfidebond with the immunoglobulin heavy chain. In some embodiments, theimmunoglobulin heavy chain is paired with an immunoglobulin light chain,so that the heavy chain variable region and the light chain variableregion form a single antigen-binding site. In some embodiments, theimmunoglobulin heavy chain is part of a single chain immunoglobulin withboth an immunoglobulin heavy chain and an immunoglobulin light chain. Insome embodiments, the heterodimeric molecule is a monovalent antibodymolecule. In some embodiments, the heterodimeric molecules expressed onthe surface of a cell do not bind a secreted antibody.

Thus, in some embodiments, a cell comprises: (a) a polynucleotideencoding for a polypeptide comprising an immunoglobulin heavy chainconstant region comprising CH2 and CH3 domains and a non-immunoglobulintransmembrane portion; and (b) at least one additional polynucleotidethat encodes at least one additional polypeptide. In some embodiments,the at least one additional polypeptide comprises an immunoglobulinheavy chain, and/or an immunoglobulin light chain. In some embodiments,the at least one additional polypeptide comprises a single chainimmunoglobulin with an immunoglobulin heavy chain and an immunoglobulinlight chain. In some embodiments, the at least one additionalpolypeptide is an antibody. In some embodiments, the additionalpolypeptide is an antibody that can be secreted from the cell. Incertain embodiments, the polypeptides of the present invention expressedon the surface of a cell do not bind a secreted antibody.

The present invention also provides hybridoma cells and methods ofproducing hybridomas, wherein the hybridoma expresses a polypeptideand/or a heterodimeric molecule described herein. In some embodiments, amethod for producing a hybridoma cell comprises fusing cells expressinga polypeptide of the present invention with an antibody-producing cell.In some embodiments, the fused hybridoma cells express a heterodimericantibody molecule on the surface of the cells. An antibody-producingcell is any cell that is capable of producing or is producing anantibody molecule including, but not limited to, a B-cell, a plasmacell, a myeloma, a hybridoma, and a recombinant cell. In someembodiments, the antibody-producing cell contributes an immunoglobulinheavy chain to the fused cells wherein the immunoglobulin heavy chainforms a heterodimeric molecule with the polypeptide expressed by thecell. In some embodiments, the antibody-producing cell contributes animmunoglobulin light chain to the fused cells, wherein theimmunoglobulin light chain associates with an immunoglobulin heavy chainto form an antigen-binding site. In some embodiments, theantibody-producing cell contributes a single chain immunoglobulin withan immunoglobulin heavy chain and an immunoglobulin light chain to thefused cells, wherein the single chain immunoglobulin forms aheterodimeric molecule with the polypeptide expressed by the cell. Insome embodiments, the antibody-producing cell is a mouse cell. In someembodiments, the antibody-producing cell is a human cell. In someembodiments, the antibody-producing cell is a population ofantibody-producing cells. In some embodiments, the antibody-producingcells are cells isolated from an immunized animal. In some embodiments,the antibody-producing cells are cells isolated from a naive animal. Inother embodiments, the antibody-producing cells comprise a plurality ofpolynucleotides. In some embodiments, the plurality of polynucleotidesencodes a plurality of polypeptides comprising immunoglobulin heavychains. In some embodiments, the plurality of polynucleotides encodes aplurality of polypeptides comprising immunoglobulin light chains. Insome embodiments, the plurality of polynucleotides encodes a pluralityof polypeptides comprising a single chain immunoglobulin with animmunoglobulin heavy chain and an immunoglobulin light chain. In someembodiments, the plurality of polynucleotides comprises a DNA library.In some embodiments, the DNA library is generated from cells of animmunized animal. In other embodiments, the DNA library is a naïvelibrary. In some embodiments, the DNA library is a cDNA library. In someembodiments, the method produces a population (e.g., a library) ofhybridoma cells that express a plurality of heterodimeric antibodymolecules. Also provided are hybridomas or hybridoma libraries producedby the methods described herein.

The present invention also provides methods of producing a cell librarythat expresses heterodimeric molecules comprising the polypeptidesdescribed herein. In some embodiments, a method of producing a celllibrary comprises transfecting cells expressing a polypeptide of thepresent invention with a plurality of polynucleotides that encode aplurality of polypeptides. In some embodiments, the transfected cellsexpress a heterodimeric molecule. In some embodiments, the heterodimericmolecule is expressed on the surface of the transfected cells. In someembodiments, the plurality of polynucleotides encodes a plurality ofpolypeptides comprising an immunoglobulin heavy chain constant regioncomprising CH2 and CH3 domains. In some embodiments, the plurality ofpolynucleotides encodes a plurality of polypeptides comprising animmunoglobulin Fc region. In some embodiments, the plurality ofpolynucleotides encodes a plurality of polypeptides comprising animmunoglobulin heavy chain. In some embodiments, the plurality ofpolynucleotides encodes a plurality of polypeptides comprising animmunoglobulin heavy chain and an immunoglobulin light chain. In someembodiments, the plurality of polynucleotides encodes a plurality ofpolypeptides comprising a single chain immunoglobulin with animmunoglobulin heavy chain and an immunoglobulin light chain. In someembodiments, the plurality of polynucleotides encodes a plurality ofpolypeptides, wherein each polypeptide comprises: (a) an immunoglobulinFc region, and (b) a randomized polypeptide. In some embodiments, theplurality of polynucleotides comprises a DNA library. In someembodiments, the DNA library is generated from cells of an immunizedanimal. In other embodiments, the DNA library is generated from cells ofa naïve animal. In some embodiments, the library is a naïve library. Insome embodiments, the DNA library is a cDNA library. In certainembodiments, the DNA library encodes for a random polypeptide library.Also provided are cell libraries made by any of the methods describedherein.

The present invention also provides cells that express the novelpolypeptides of the present invention and also produce antibodies. Alsoprovided are methods of producing these cells, wherein the cells expressa polypeptide of the present invention, an antibody, and/or aheterodimeric molecule as described herein. In some embodiments, anantibody-producing cell is transfected with a polynucleotide thatencodes any of the novel polypeptides described herein. In someembodiments, the transfected cells express the polypeptide. In someembodiments, the transfected cells express a heterodimeric molecule onthe surface of the cells. In some embodiments, the heterodimericmolecule comprises the polypeptide and an immunoglobulin heavy chain. Insome embodiments, the immunoglobulin heavy chain is associated with animmunoglobulin light chain to form a single antigen-binding site. Insome embodiments, the immunoglobulin heavy chain is part of a singlechain immunoglobulin. In some embodiments, the antibody-producing cellis a hybridoma library. In some embodiments, the antibody-producing cellis a hybridoma.

The present invention also provides cell libraries comprising the novelpolypeptides as described herein. In some embodiments, each cell in acell library comprises: (a) a first polypeptide comprising animmunoglobulin heavy chain constant region comprising CH2 and CH3domains; and a transmembrane portion; and (b) a second polypeptidecomprising an immunoglobulin heavy chain, wherein the two polypeptidesare able to form a heterodimeric molecule. In some embodiments, the twopolypeptides covalently associate. In some embodiments, the twopolypeptides form at least one disulfide bond. In some embodiments, theheterodimeric molecule is expressed on the surface of the cells. In someembodiments, each cell further comprises an immunoglobulin light chain.In some embodiments, the second polypeptide is a single chainimmunoglobulin. In some embodiments, the heterodimeric moleculecomprises a single antigen-binding site.

The present invention also provides methods of identifying and/orselecting polypeptides that are not antibodies. The polypeptides caninclude, but are not limited to, cell surface receptors or fragmentsthereof, soluble receptors or fragments thereof, cell surface ligands orfragments thereof, and soluble ligands or fragments thereof. Thepolypeptides can include mutagenized or derivatized versions ofproteins. The polypeptides can include randomized polypeptides, forexample a randomized polypeptide library.

In some embodiments, each cell in a cell library comprises: (a) a firstpolypeptide comprising an immunoglobulin heavy chain constant regioncomprising CH2 and CH3 domains; and a transmembrane portion; and (b) asecond polypeptide. In some embodiments, the two polypeptides covalentlyassociate. In some embodiments, the two polypeptides form at least onedisulfide bond. In some embodiments, the second polypeptide comprises animmunoglobulin heavy chain constant region comprising CH2 and CH3domains, wherein the two polypeptides are able to form a heterodimericmolecule. In some embodiments, the second polypeptide comprises animmunoglobulin Fc region. In some embodiments, the second polypeptidecomprises a single chain immunoglobulin with an immunoglobulin heavychain and an immunoglobulin light chain. In some embodiments, the secondpolypeptide comprises a randomized polypeptide. In some embodiments, thesecond polypeptide comprises: (a) an immunoglobulin Fc region and (b) arandomized polypeptide.

Randomized polypeptides or a random polypeptide library (and thepolynucleotides encoding for them) can be either fully randomized orthey can be biased in their randomization. Any method known to those ofskill in the art can be used to generate randomized polypeptides or arandom peptide library, including but not limited to, chemical synthesisof the nucleotides and/or peptides. (See e.g., US Patent Appl. No.2003/0170753 and International Appl. No. WO 00/20574).

The cell libraries described herein can be screened for cells producinga specific polypeptide. Thus, provided herein are methods of screening acell library comprising contacting a cell library described herein witha detection molecule. In some embodiments, the method further comprisesidentifying the cells that are bound by the detection molecule. In someembodiments, the method further comprises isolating the cells that arebound by the detection molecule. The detection molecule can include, butis not limited to, proteins, carbohydrates, small molecules, andvariants thereof. In some embodiments, the detection molecule is aprotein or fragment thereof. In some embodiments, the detection moleculeis an antigen of interest. In some embodiments, the detection moleculeis a cell surface receptor, an antibody, a ligand or fragments thereof.In some embodiments, the detection molecule is labeled. In someembodiments, the detection molecule is labeled with a fluorophore, achromophore or a magnetic compound. In some embodiments, the cells boundby the detection molecule are identified by flow cytometry. In someembodiments, the cells hound by the detection molecule are isolated byFACS.

IV. Methods of Identifying, Selecting, and/or Isolating Antibodies orPolypeptides

The identification of hybridoma cells that produce monoclonal antibodiesto an antigen of interest is typically accomplished by ELISA screeningof cell supernatants. The supernatants can be produced by random cellsisolated by single cell (limiting dilution) cloning techniques. Or thesupernatants can be produced by pools of hybridoma cells from ahybridoma library, whereby the cells from any ELISA-positive pool mustbe sub-cloned and isolated by single cell cloning. These processes havemany limitations. In the first case, large numbers of plates containing“single cell” cultures must be screened to find ELISA-positive clones.In the case of hybridoma pool screening, each ELISA-positive pool mustbe further separated by limiting dilution cloning to isolate a pureclone of the hybridoma of interest. Both of these methods are laborintensive and time consuming. Furthermore, in some circumstances thedesired clone may represent an extremely low percentage of the hybridomalibrary, making the identification of the rare clone difficult. Inaddition, the ELISA screening approach identifies binding activity to asingle antigen. To assess whether an individual hybridoma clone isproducing a monoclonal antibody able to bind to more than one antigen,multiple, sequential ELISAs need to be performed.

The novel polypeptides, cells, and cell libraries of the presentinvention can be used in methods of identifying and isolating a cellproducing an antibody specific for an antigen or target of interest. Insome embodiments, a method of identifying a cell that produces aspecific antibody comprises fusing cells comprising a novel polypeptideas described herein, with an antibody-producing cell to produce apopulation of hybridoma cells. In some embodiments, the hybridoma cellsexpress a heterodimeric antibody molecule on the surface of the cells.In some embodiments, the method comprises contacting the population ofhybridoma cells with a detection molecule. In some embodiments, themethod comprises identifying the hybridoma cells that are bound by thedetection molecule. In some embodiments, the method comprises isolatingthe cells that are bound by the detection molecule. In some embodiments,the detection molecule is a target of interest. In some embodiments, thedetection molecule is a protein or fragment thereof. In someembodiments, the detection molecule is labeled.

In some embodiments, hybridoma cells producing an antibody molecule areidentified using a form of affinity selection known to those of skill inthe art as “panning”. The cells are incubated with a detection molecule(e.g., an antigen of interest) and cells identified by the detectionmolecule are isolated. Immunoglobulin DNA from the isolated cells isamplified and reintroduced into cells. The cells are incubated againwith the detection molecule and cells bound by the detection moleculeare isolated. One or more rounds of selection can enrich for antibodiesor fragments thereof with the desired specificity to the target ofinterest. Thus, rare cells expressing a desired antibody molecule can beselected from a large, highly diverse population.

As described herein, in some embodiments the polypeptide of theinvention comprises an immunoglobulin heavy chain constant regioncomprising CH2 and CH3 and a transmembrane portion. The polypeptide iscapable of covalently associating with an immunoglobulin heavychain-light chain pair expressed by the antibody-producing cell to forma heterodimeric antibody molecule expressed on the surface of the cell.In some embodiments, the heterodimeric antibody molecule comprises asingle antigen-binding site and is a monovalent antibody. The singleantigen-binding site of the heterodimeric molecule on an individual cellis representative of the binding specificity of the antibody producedand secreted by the individual cell. Thus identifying a cell with aheterodimeric antibody molecule that binds to a specific antigen ofinterest, allows for isolation of the cell which also produces asecreted antibody with the same binding specificity.

The antibody-producing cell can be any cell that is producing anantibody, whether the cell naturally produces an antibody (e.g., aB-cell) or whether the cell makes an antibody by recombinant means(e.g., a transfected cell). Thus, in some embodiments, theantibody-producing cell is a B-cell, a plasma cell, a hybridoma, amyeloma, or a recombinant cell. In some embodiments, theantibody-producing cell is a mouse cell. In some embodiments, theantibody-producing cell is a human cell. In some embodiments, theantibody-producing cell is a population of antibody-producing cells. Insome embodiments, the antibody-producing cells are from an immunizedanimal. In some embodiments, the antibody-producing cells are from anaïve animal.

In some embodiments, the antibody-producing cells comprise a pluralityof polynucleotides. In some embodiments, the plurality ofpolynucleotides encodes a plurality of polypeptides comprisingimmunoglobulin heavy chains. In some embodiments, the plurality ofpolynucleotides encodes a plurality of polypeptides further comprisingimmunoglobulin light chains. In some embodiments, the plurality ofpolynucleotides encodes a plurality of polypeptides comprising a singlechain immunoglobulin with an immunoglobulin heavy chain and animmunoglobulin light chain. In some embodiments, the plurality ofpolynucleotides comprises a DNA library. In some embodiments, the DNAlibrary is generated from cells of an immunized animal. In someembodiments, the DNA library is generated from cells of a naïve animal.In some embodiments, the DNA library is a naïve library. In otherembodiments, the DNA library is a cDNA library.

The methods provided herein do not limit the type of antibody that isproduced by the antibody-producing cells. Thus, in some embodiments, theantibody made by the antibody-producing cell is a recombinant antibody,a monoclonal antibody, a chimeric antibody, a humanized antibody, ahuman antibody, a single chain antibody, or an antibody fragment. Insome embodiments, the antibody made by the antibody-producing cell is amonospecific antibody or a multispecific antibody (e.g., a bispecificantibody). In some embodiments, the antibody made by theantibody-producing cell is an IgA, IgD, IgE, IgG or IgM antibody or asubtype thereof. In some embodiments, the antibody made by theantibody-producing cell is an IgG 1 or IgG2 antibody. In someembodiments, the antibody made by the antibody-producing cell is an IgG2antibody. In some embodiments, the antibody is a murine antibody. Insome embodiments, the antibody made by the antibody-producing cell is ahumanized antibody. In other embodiments, the antibody is a humanantibody.

In some embodiments, the method of identifying a cell that produces aspecific antibody comprises transfecting cells comprising a novelpolypeptide described herein with at least one polynucleotide encodingat least one additional polypeptide. In some embodiments, thetransfected cells express a heterodimeric antibody molecule on thesurface of the cells. In some embodiments, the method comprisescontacting the transfected cells with a detection molecule. In someembodiments, the method comprises identifying the transfected cells thatare bound by the detection molecule. In some embodiments, the methodcomprises isolating the cells that bound by the detection molecule. Insome embodiments, the detection molecule is a target of interest. Insome embodiments, the detection molecule is a protein or fragmentthereof. In some embodiments, the detection molecule is labeled.

In some embodiments, the at least one polynucleotide comprises aplurality of polynucleotides that encode a plurality of polypeptides. Insome embodiments, the plurality of polynucleotides encodes a pluralityof polypeptides comprising an immunoglobulin heavy chain. In someembodiments, the plurality of polynucleotides encodes a plurality ofpolypeptides further comprising an immunoglobulin light chain. In someembodiments, the plurality of polynucleotides encodes a plurality ofpolypeptides comprising a single chain immunoglobulin with animmunoglobulin heavy chain and an immunoglobulin light chain. In someembodiments, the plurality of polynucleotides comprises a DNA library.In some embodiments, the DNA library is generated from cells of animmunized animal. In some embodiments, the DNA library is generated fromcells of a naïve animal. In some embodiments, the DNA library is a naïvelibrary. In other embodiments, the DNA library is a cDNA library.

In some embodiments, the at least one polynucleotide encodes apolypeptide that is a recombinant antibody, a monoclonal antibody, achimeric antibody, a humanized antibody, a human antibody, a singlechain antibody, or an antibody fragment. In some embodiments, the atleast one polynucleotide encodes a polypeptide that is a monospecificantibody or a multispecific antibody (e.g., a bispecific antibody). Insome embodiments, the at least one polynucleotide encodes a polypeptidethat is an IgA, IgD, IgE, IgG or IgM antibody or a subtype thereof insome embodiments, the at least one polynucleotide encodes a polypeptidethat is an IgG1 or IgG2 antibody. In some embodiments, the at least onepolynucleotide encodes a polypeptide that is an IgG2 antibody. In someembodiments, the at least one polynucleotide encodes a polypeptide thatis a murine antibody. In some embodiments, the at least onepolynucleotide encodes a polypeptide that is a humanized antibody. Inother embodiments, the at least one polynucleotide encodes a polypeptidethat is a human antibody.

In some embodiments, the method of identifying a cell that produces aspecific antibody comprises transfecting a cell library with apolynucleotide or vector encoding any of the novel polypeptidesdescribed herein, wherein the cell library comprises antibody-producingcells. In some embodiments, the transfected cells express aheterodimeric antibody molecule on the surface of the cells. In someembodiments, the method comprises contacting the transfected cells witha detection molecule. In some embodiments, the method comprisesidentifying the transfected WIN that are bound by the detectionmolecule. In some embodiments, the method comprises isolating the cellsthat bound by the detection molecule. In some embodiments, the detectionmolecule is a target of interest. In some embodiments, the detectionmolecule is a protein or fragment thereof. In some embodiments, thedetection molecule is labeled.

In some embodiments, the cell library is B-cells, plasma cells,hybridomas, myelomas, or recombinant cells. In some embodiments, thecell library is a hybridoma library. In some embodiments, the celllibrary comprises mouse cells. In some embodiments, the cell librarycomprises human cells.

In some embodiments, the methods provided herein produce cells orhybridomas that secrete a specific antibody but also express on theircell surface a monovalent heterodimeric antibody molecule that containsa single antigen-binding site representative of the binding specificityof the secreted antibody. This heterodimeric antibody molecule, andhence the antibody-producing cell, can be identified using a detectionmolecule. In some embodiments, the detection molecule is an antigen ofinterest. In some embodiments, the detection molecule is a protein orfragment thereof. In some embodiments, the detection molecule islabeled. In some embodiments, the detection molecule is labeled with afluorophore, a chromophore or a magnetic compound. In some embodiments,the cells bound with the detection molecule are identified by flowcytometry. In some embodiments, the cells bound with the detectionmolecule are isolated by FACS.

The novel polypeptides, cells, and cell libraries of the presentinvention can be used in methods of screening for an antibody specificfor an antigen or target of interest. In some embodiments, a method ofscreening for a specific antibody comprises screening the cells or celllibraries that are described herein. In some embodiments, the cells(e.g., cell library) express a heterodimeric molecule on the surface ofthe cells. As described herein, the heterodimeric molecule expressed onthe surface of the cell comprises a binding site that is the same as thebinding site of an antibody produced by the cell. In some embodiments,the method of screening for a specific antibody comprises contacting thecells with a detection molecule. The cells expressing a heterodimericmolecule bound by the detection molecule express an antibody moleculespecific for the detection molecule. In some embodiments, the method ofscreening comprises identifying the cells that are bound by thedetection molecule. In some embodiments, the method of screeningcomprises isolating the cells that are bound by the detection molecule.In some embodiments, the method of screening comprises isolating theantibody produced by the cells that are bound by the detection molecule.In some embodiments, the detection molecule is a target of interest. Insome embodiments, the detection molecule is a protein or fragmentthereof. In some embodiments, the detection molecule is labeled.

In some embodiments, screening for specific antibodies using theMembrane-MAb technique involves iterative rounds of selection followedby amplification. For example, cells (e.g., 293-hMT cells or 293T-hMTcells) are transfected with library DNA. The amount of DNA and thenumber of cells used for any one transfection will depend on the libraryand/or library diversity. Once transfected, cells are incubated andallowed to grow for 24-48 hours. Cells are then harvested, washed, andscreened with a target molecule (e.g., an antigen of interest). Thescreening molecule may be directly labeled or may be detected by asecond molecule which is labeled. The labeled cells are analyzed and/orsorted using methods known to one of skill in the art, for example, byFACS or by using magnetic beads. Plasmid DNA from the sorted cells isextracted and amplified. Plasmid DNA may be extracted and purified byany well-known method. For example, plasmid DNA can be extracted using aphenol/chloroform mixture and then ethanol precipitated. The purifiedplasmid DNA is then used to transform bacteria and amplify the isolatedDNA. Or DNA may be isolated from the cells and specific regionsamplified by PCR methods known to those of skill in the art. PCRproducts are subcloned back into the host plasmid which is used totransform bacteria and amplify the plasmid. Using either method, theresulting amplified library output from the first selection is used totransfect fresh cells (e.g., 293-hMT or 293T-hMT cells) and a subsequentround of selection with the same target or antigen is performed. In thismanner multiple rounds of selection are performed until the populationof antibodies that specifically bind the target or antigen of interestis enriched.

Once the library has been enriched for a population of specific antibodymolecules, DNA is isolated and used to transform bacteria. Singlecolonies are picked and plasmid DNA is isolated. The clonal plasmids areused to transfect mammalian cells (e.g., parental HEK-293 or parental293T cells) for the production of soluble antibody. The resultingantibody is then tested for specific binding to the desired target byELISA, FACS and/or Biacore.

Transient transfection of mammalian cells with a plasmid generallyresults in multiple copies of plasmid per cell. The copy number per cellcan range from 100-10,000. The absolute number per cell depends on avariety of factors including, but not limited to, transfection protocol,transfection reagents, plasmid quality, plasmid size, and cellulardensity. In generating libraries of cell surface-displayed antibodies itis desirable to modulate the amount of antibody-encoding plasmid (or Ablibrary plasmid) introduced into the cell. In some embodiments, tomodulate the plasmid copy number of Ab library plasmid in cells, aseparate “carrier” plasmid may be used. The carrier plasmid is mixedwith the Ab library plasmid and may take up some of the available“plasmid space”. Thus in some embodiments, to modulate the number of Ablibrary plasmids taken up by the cells, carrier plasmid is mixed withthe Ab library plasmid at different ratios and then transfected intocells.

In some embodiments, a method of modulating expression of a polypeptideon the surface of a host cell comprises transfecting into a host cell(a) DNA encoding a polypeptide, and (b) an excess amount of irrelevantDNA. In some embodiments, the polypeptide is a heterodimeric antibodymolecule. Thus in some embodiments, a method of modulating expression ofa heterodimeric antibody molecule on the surface of a host cellcomprises transfecting into a host cell (a) DNA encoding animmunoglobulin, and (b) an excess amount of irrelevant DNA. In someembodiments, the ratio of DNA encoding an immunoglobulin to irrelevantDNA is from 1:10 to 1:1,000,000. In some embodiments, the ratio of DNAencoding an immunoglobulin to irrelevant DNA is 1:10, 1:100, 1:1000,1:10,000, 1:100,000, 1:1,000,000, or any ratio in between. In someembodiments, the DNA encoding an immunoglobulin is plasmid DNA and theirrelevant DNA is plasmid DNA. In some embodiments, the host cellcomprises any of the polypeptides or polynucleotides described herein.In some embodiments, the immunoglobulin comprises an immunoglobulinheavy chain. In some embodiments, the antibody comprises animmunoglobulin light chain. In some embodiments, the immunoglobulin is asingle chain antibody molecule with an immunoglobulin heavy chain and animmunoglobulin light chain. In some embodiments, the DNA encoding animmunoglobulin is a DNA library. In some embodiments, the DNA library isgenerated from cells of an immunized animal. In some embodiments, theDNA library is generated from cells of a naïve animal. In someembodiments, the DNA library is a cDNA library. In some embodiments, themethod further comprises contacting the host cell with a detectionmolecule. In some embodiments, the method comprises identifying the hostcells that are bound by the detection molecule. In some embodiments, themethod comprises isolating the host cells that are bound by thedetection molecule.

Also provided in the present invention are antibodies produced by a cellidentified, selected, and/or isolated by any of the methods describedherein. Also provided in the present invention are antibodies producedby a cell isolated by any of the methods described herein.

V. Antibodies

As described herein the novel polypeptides and cells of the presentinvention may be used to identify, select, and/or isolate antibodiesthat specifically bind targets (e.g., antigens) of interest. In someembodiments, the heterodimeric antibody molecules expressed on thesurface of the cells comprise an immunoglobulin heavy chain-light chainpair that forms a single antigen-binding site. The singleantigen-binding site is identical to, and/or representative of, theantigen-binding sites on the antibodies produced and secreted by thecells.

The polypeptides, cells and methods provided herein do not limit thetype of antibody that is produced. In some embodiments, the antibodiesare monoclonal antibodies. Monoclonal antibodies can be prepared usinghybridoma methods known to one of skill in the art (see e.g., Kohler andMilstein, 1975, Nature 256:495). In some embodiments, a mouse, hamster,or other appropriate host animal, is immunized by multiple subcutaneous,intraperitoneal or intravenous injections of the relevant antigen (e.g.,a purified peptide fragment, full-length recombinant protein, fusionprotein, etc.). The antigen can be optionally conjugated to a carrierprotein such as keyhole limpet hemocyanin (KLH) or serum albumin. Theantigen (with or without a carrier protein) is diluted in sterile salineand usually combined with an adjuvant (e.g., Complete or IncompleteFreund's Adjuvant) to form a stable emulsion. In some embodiments, theimmunizing antigen can be a human protein or a portion thereof. In someembodiments, the immunizing antigen can be a mouse protein or a portionthereof. In some embodiments, a mouse is immunized with a human antigen.In some embodiments, a mouse is immunized with a mouse antigen. In someembodiments, isolated lymphocytes can be immunized in vitro. In someembodiments, the isolated lymphocytes are non-specifically activated. Insome embodiments, the isolated lymphocytes are mouse lymphocytes. Insome embodiments, the isolated lymphocytes are human lymphocytes.

Following immunization and/or activation, lymphocytes are isolated andfused with a suitable cell line using, for example, polyethylene glycolto produce hybridomas. The cell line used to produce a hybridoma mayinclude any of the cell lines expressing a novel polypeptide describedherein. In some embodiments, the hybridoma cells are selected usingspecialized media as known in the art and unfused lymphocytes and fusioncells do not survive the selection process. In some embodiments,hybridomas that produce monoclonal antibodies directed against a chosenantigen may be identified by a variety of techniques including, but notlimited to, immunoprecipitation, immunoblotting, and in vitro bindingassays (e.g., flow cytometry (FACS), enzyme-linked immunosorbent assay(ELISA), radioimmunoassay (RIA)). In other embodiments, the cells aredirectly selected based upon the antigen binding site expressed on thesurface of the cells as part of a heterodimeric antibody molecule usingany of the methods as described herein. Hybridomas can be propagatedeither in in vitro culture using standard methods (God: g, MonoclonalAntibodies: Principles and Practice, Academic Press, 1986) or in in vivoas ascites in an animal. The monoclonal antibodies can be purified fromthe culture medium or ascites fluid according to standard methods in theart including, but not limited to, affinity chromatography, ion-exchangechromatography, gel electrophoresis, and dialysis.

Alternatively, monoclonal antibodies can be made using recombinant DNAtechniques as known to one skilled in the art (see e.g., U.S. Pat. No.4,816,567). In some embodiments, polynucleotides encoding a monoclonalantibody are isolated from mature 13-cells or hybridoma cells, usingtechniques such as RT-PCR with oligonucleotide primers that specificallyamplify the genes encoding the heavy and light chains of antibodies. Thesequences of the isolated polynucleotides can be determined usingconventional sequencing techniques. The isolated polynucleotidesencoding the heavy and light chains can be cloned into suitableexpression vectors. These expression vectors produce the monoclonalantibodies when transfected into host cells such as E. coli, simian COScells, Chinese hamster ovary (CHO) cells, human embryonic kidney cells(HEK), or myeloma Bells that do not otherwise produce immunoglobulinproteins. In some embodiments, isolated polynucleotides encoding heavyand light chains are transfected into the cells of the present inventionthat express the polypeptide comprising an immunoglobulin heavy chainregion comprising CH2 and CH3 domains and a transmembrane portion.

Recombinant monoclonal antibodies, or fragments thereof, can also beisolated from phage display libraries expressing CDRs of the desiredspecies (see e.g., McCafferty et al., 1990, Nature, 348:552-554;Clackson et al., 1991, Nature, 352:624-628; and Marks et al., 1991, J.Biol., 222:581-597).

The polynucleotide(s) encoding a monoclonal antibody can be furthermodified using recombinant DNA technology to generate alternativeantibodies. In some embodiments, the constant domains of the light andheavy chains of, for example, a mouse monoclonal antibody can besubstituted 1) for those regions of, for example, a human antibody togenerate a chimeric antibody or 2) for a non-immunoglobulin polypeptideto generate a fusion antibody. In some embodiments, the constant regionsare truncated or removed to generate the desired antibody fragment of amonoclonal antibody. Site-directed or high-density mutagenesis of thevariable region can be used to optimize specificity, affinity, and/orother biological characteristics of a monoclonal antibody. In someembodiments, site-directed mutagenesis of the CDRs can be used tooptimize specificity, affinity, and/or other biological characteristicsof a monoclonal antibody.

In some embodiments, the antibody is a humanized antibody. Typically,humanized antibodies are human immunoglobulins in which residues fromthe complementary determining regions (CDRs) are replaced by residuesfrom CDRs of a non-human species (e.g., mouse, rat, rabbit, hamster)that have the desired specificity, affinity, and/or capability usingmethods known to one skilled in the art. In some embodiments, the Fvframework region (FR) residues of a human immunoglobulin are replacedwith the corresponding framework region residues from a non-humanimmunoglobulin that has the desired specificity, affinity, and/orcapability. In some embodiments, the humanized antibody can be furthermodified by the substitution of additional residues either in the Fvframework region and/or within the replaced non-human residues to refineand optimize antibody specificity, affinity, and/or capability. Ingeneral, the humanized antibody will comprise substantially all of atleast one, and typically two or three, variable domains containing all,or substantially all, of the CDRs that correspond to the non-humanimmunoglobulin whereas all, or substantially all, of the frameworkregions are those of a human immunoglobulin consensus sequence. In someembodiments, the humanized antibody can also comprise at least a portionof an immunoglobulin constant region or domain (Fc), typically that of ahuman immunoglobulin. In certain embodiments, such humanized antibodiesare used therapeutically because they may reduce antigenicity and HAMA(human anti-mouse antibody) responses when administered to a humansubject. One skilled in the art would be able to obtain a functionalhumanized antibody with reduced immunogenicity following knowntechniques (see for example U.S. Pat. Nos. 5,225,539; 5,585,089;5,693,761; and 5,693,762).

In certain embodiments, the antibody is a human antibody. Humanantibodies can be directly prepared using various techniques known inthe art. Immortalized human B lymphocytes, immunized in vitro orisolated from an immunized individual, that produce an antibody directedagainst a target antigen can be generated. Alternatively, a humanantibody can be selected from a phage library, where that phage libraryexpresses human antibodies (see e.g., Vaughan et al., 1996, Nat.Biotech., 14:309-314; Sheets et al., 1998, PNAS, 95:6157-6162;Hoogenboom and Winter, 1991, J. Mol. Biol., 227:381; and Marks et al.,1991, J. Mol. Biol., 222:581). Techniques for the generation and use ofantibody phage libraries are also described in U.S. Pat. Nos. 5,969,108;6,172,197; 5,885,793; 6,521,404; 6,544,731; 6,555,313; 6,582,915;6,593,081; 6,300,064; 6,653,068; 6,706,484; and 7,264,963; and Rothe etal., 2008, J. Mol. Bio., 376:1182-1200. Affinity maturation strategiesand chain shuffling strategies are known in the art and may be employedto generate high affinity human antibodies. (Marks et al., 1992,Bio/Technology, 10:779-783).

Human antibodies can also be made in transgenic mice containing humanimmunoglobulin loci that are capable, upon immunization, of producingthe full repertoire of human antibodies in the absence of endogenousimmunoglobulin production. This approach is described in U.S. Pat. Nos.5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016.

In certain embodiments, the antibody is a bispecific antibody.Bispecific antibodies are capable of specifically recognizing andbinding to at least two different epitopes. The different epitopes caneither be within the same molecule or on different molecules. In someembodiments, the antibodies can specifically recognize and bind a firstantigen target, as well as a second antigen target, such as an effectormolecule on a leukocyte (e.g., CD2, CD3, CD28, or B7) or a Fc receptor(e.g., CD64, CD32, or CD 16) so as to focus cellular defense mechanismsto the cell expressing the first antigen target. In some embodiments,the antibodies can be used to direct cytotoxic agents to cells thatexpress a particular target antigen. These antibodies possess anantigen-binding arm and an arm that binds a cytotoxic agent or aradionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA.

Techniques for making bispecific antibodies are known by those skilledin the art, see for example, Millstein et al., 1983, Nature,305:537-539; Brennan et al., 1985, Science, 229:81; Suresh et al., 1986,Methods in Enzymol., 121:120; Traunecker et al., 1991, EMBO J.,10:3655-3659; Shalaby et al., 1992, J. Exp. Med., 175:217-225; Kostelnyet al., 1992, J. Immunol., 148:1547-1553; Gruber et al., 1994, J.Immunol., 152:5368; and U.S. Pat. No. 5,731,168). Bispecific antibodiescan be intact antibodies or antibody fragments. Antibodies with morethan two valencies are also contemplated. For example, trispecificantibodies can be prepared (Tutt et al., 1991, J. Immunol., 147:60).Thus, in certain embodiments the antibodies are multispecific.

In some embodiments, the antibody describe herein is a single chainimmunoglobulin produced from a single gene construct (see e.g., Lee etal., 1999, Molecular Immunology, 36:61-71). The single chainimmunoglobulin contains both an immunoglobulin heavy chain and animmunoglobulin light chain in their entirety. For example, the carboxylend of a light chain is joined, via a Gly-Ser linker peptide, to theamino end of a heavy chain, wherein the light chain comprises a variableregion and a constant region and the heavy chain comprises a variableregion and CH1, CH2 and CH3. These single chain immunoglobulins formdimeric antibody molecules.

In certain embodiments, the antibodies or other polypeptides describedherein may be monospecific.

In certain embodiments, the antibody is an antibody fragment. Antibodyfragments may have different functions or capabilities than intactantibodies; for example, antibody fragments can have increased tumorpenetration. Various techniques are known for the production of antibodyfragments including, but not limited to, proteolytic digestion of intactantibodies. In some embodiments, antibody fragments include a F(ab′)2fragment produced by pepsin digestion of an antibody molecule. In someembodiments, antibody fragments include a Fab fragment generated byreducing the disulfide bridges of an F(ab′)2 fragment. In otherembodiments, antibody fragments include a Fab fragment generated by thetreatment of the antibody molecule with papain and a reducing agent. Incertain embodiments, antibody fragments are produced recombinantly. Insome embodiments, antibody fragments include Fv or single chain Fv(scFv) fragments. Fab, Fv, and scFv antibody fragments can be expressedin, and secreted from, E. coli or other host cells, allowing for theproduction of large amounts of these fragments. In some embodiments,antibody fragments are isolated from antibody phage libraries asdiscussed herein. For example, methods can be used for the constructionof Fab expression libraries (Huse et al., 1989, Science, 246:1275-1281)to allow rapid and effective identification of monoclonal Fab fragmentswith the desired specificity for a protein or derivatives, fragments,analogs or homologs thereof. In some embodiments, antibody fragments arelinear antibody fragments as described in U.S. Pat. No. 5,641,870. Incertain embodiments, antibody fragments are monospecific or bispecific.In certain embodiments, the antibody is a scFv. Various techniques canbe used for the production of single-chain antibodies specific to agiven target antigen (see, e.g., U.S. Pat. No. 4,946,778).

It can farther be desirable, especially in the case of antibodyfragments, to modify an antibody in order to increase its serumhalf-life. This can be achieved, for example, by incorporation of asalvage receptor binding epitope into the antibody fragment by mutationof the appropriate region in the antibody fragment or by incorporatingthe epitope into a peptide tag that is then fused to the antibodyfragment at either end or in the middle (e.g., by DNA or peptidesynthesis). (See e.g., U.S. Pat. Nos. 6,096,871 and 6,121,022.)

For the purposes of the present invention, it should be appreciated thatmodified antibodies, or fragments thereof, can comprise any type ofvariable region that provides for the association of the antibody withthe specific antigen its binds. In this regard, the variable region maybe derived from any type of mammal that can be induced to mount ahumoral response and generate immunoglobulins against a desired antigen.As such, the variable region of the modified antibodies can be, forexample, of human, murine, non-human primate (e.g., cynomolgus monkeys,macaques, etc.) or rabbit origin. In some embodiments, both the variableand constant regions of the modified immunoglobulins are human. In otherembodiments, the variable regions of compatible antibodies (usuallyderived from a non-human source) can be engineered or specificallytailored to improve the binding properties or reduce the immunogenicityof the molecule. In this respect, variable regions useful in the presentinvention can be humanized or otherwise altered through the inclusion ofimported amino acid sequences.

In certain embodiments, the variable domains in both the heavy and lightchains are altered by at least partial replacement of one or more CDRsand, if necessary, by partial framework region replacement and sequencemodification. Although the CDRs may be derived from an antibody of thesame class or even subclass as the antibody from which the frameworkregions are derived, it is envisaged that the CDRs will be derived froman antibody of a different class and preferably from an antibody from adifferent species. It may not be necessary to replace all of the CDRswith all of the CDRs from the donor variable region to transfer theantigen binding capacity of one variable domain to another. Rather, itmay only be necessary to transfer those residues that are necessary tomaintain the activity of the antigen binding site.

Alterations to the variable region notwithstanding, those ski/led in theart will appreciate that the modified antibodies of this invention willcomprise antibodies (e.g., full-length antibodies or antigen-bindingfragments thereof) in which at least a fraction of one or more of theconstant region domains has been deleted or otherwise altered so as toprovide desired biochemical characteristics, such as increased tumorlocalization, increased tumor penetration, reduced serum half-life, orincreased serum half-life, when compared with an antibody ofapproximately the same immunogenicity comprising a native or unalteredconstant region. In some embodiments, the constant region of themodified antibodies comprises a human constant region. Modifications tothe constant region include additions, deletions or substitutions of oneor more amino acids in one or more domains. The modified antibodiesdisclosed herein may comprise alterations or modifications to one ormore of the three heavy chain constant domains (CH1, CH2 or CH3) and/orto the light chain constant domain (CL). In some embodiments, one ormore domains are partially or entirely deleted from the constant regionsof the modified antibodies. In some embodiments, the entire CH2 domainhas been removed (ΔCH2 constructs). In some embodiments, the omittedconstant region domain is replaced by a short amino acid spacer (e.g.,10 aa residues) that provides some of the molecular flexibilitytypically imparted by the absent constant region.

In certain embodiments, the modified antibodies are engineered to fusethe CH3 domain directly to the hinge region of the antibody. In otherembodiments, a peptide spacer is inserted between the hinge region andthe modified CH2 and/or CH3 domains. For example, constructs may beexpressed wherein the CH2 domain has been deleted and the remaining CH3domain (modified or unmodified) is joined to the hinge region with a5-20 amino acid spacer. Such a spacer may be added to ensure that theregulatory elements of the constant domain remain free and accessible orthat the hinge region remains flexible. However, it should be noted thatamino acid spacers can, in some cases, prove to be immunogenic andelicit an unwanted immune response against the construct. Accordingly,in certain embodiments, any spacer added to the construct will berelatively non-immunogenic so as to maintain the desired biologicalqualities of the modified antibodies.

In some embodiments, the modified antibodies may have only a partialdeletion of a constant domain or substitution of a few or even a singleamino acid. For example, the mutation of a single amino acid in selectedareas of the CH2 domain may be enough to substantially reduce Fc bindingand thereby increase tumor localization and/or tumor penetration.Similarly, it may be desirable to simply delete a part of one or moreconstant region domains that control a specific effector function (e.g.,complement C1q binding) to be modulated. Such partial deletions of theconstant regions may improve selected characteristics of the antibody(serum half-life) while leaving other desirable functions associatedwith the subject constant region domain intact. Moreover, as alluded toabove, the constant regions of the disclosed antibodies may be modifiedthrough the mutation or substitution of one or more amino acids thatenhances the profile of the resulting construct. In this respect it maybe possible to disrupt the activity provided by a conserved binding site(e.g., Fe binding) while substantially maintaining the configuration andimmunogenic profile of the modified antibody. In certain embodiments,the modified antibodies comprise the addition of one or more amino acidsto the constant region to enhance desirable characteristics such asdecreasing or increasing effector function or provide for more cytotoxinor carbohydrate attachment sites.

It is known in the art that the constant region mediates severaleffector functions. For example, binding of the C1 component ofcomplement to the Fc region of IgG or IgM antibodies (bound to antigen)activates the complement system. Activation of complement is importantin the opsonization and lysis of cell pathogens. The activation ofcomplement also stimulates the inflammatory response and can also beinvolved in autoimmune hypersensitivity. In addition, the Fc region ofan antibody can bind to a cell expressing a Fc receptor (FcR). There area number of Fc receptors that are specific for different classes ofantibody, including IgG (gamma receptors), IgE (epsilon receptors), IgA(alpha receptors) and IgM (mu receptors). Binding of antibody to Fcreceptors on cell surfaces triggers a number of important and diversebiological responses including engulfment and destruction ofantibody-coated particles, clearance of immune complexes, lysis ofantibody-coated target cells by killer cells (ADCC), release ofinflammatory mediators, placental transfer and control of immunoglobulinproduction.

In certain embodiments, the antibodies provide for altered effectorfunctions that, in turn, affect the biological profile of theadministered antibody. For example, in some embodiments, the deletion orinactivation (through point mutations or other means) of a constantregion domain may reduce Fc receptor binding of the circulating modifiedantibody thereby increasing tumor localization and/or penetration. Inother embodiments, the constant region modifications increase or reducethe serum half-life of the antibody. In some embodiments, the constantregion is modified to eliminate disulfide linkages or oligosaccharidemoieties allowing for enhanced tumor localization and/or penetration.

In certain embodiments, an antibody does not have one or more effectorfunctions. In some embodiments, the antibody has no antibody-dependentcellular cytoxicity (ADCC) activity and/or no complement-dependentcytoxicity (CDC) activity. In certain embodiments, the antibody does notbind to an Fc receptor and/or complement factors. In certainembodiments, the antibody has no effector function.

The present invention further embraces variants and equivalents that aresubstantially homologous to the chimeric, humanized and humanantibodies, or antibody fragments thereof, set forth herein. These cancontain, for example, conservative substitution mutations, i.e. thesubstitution of one or more amino acids by similar amino acids.

EXAMPLES Example 1 Generation of Immunoglobulin Constant Region-CD4Constructs

A construct was designed to contain a signal sequence, a FLAG epitopetag, the hinge region and CH2 and CH3 domains of murine IgG 1 and thetransmembrane domain (TM) and intracellular domain (ICD) of human CD4,referred to as Membrane-MAb(mIgG1). A second construct was designed tocontain a signal sequence, a FLAG epitope tag, the hinge region and CH2and CH3 domains of human IgG2 and the transmembrane domain (TM) andintracellular domain (ICD) of human CD4, referred to asMembrane-MAb(hIgG2). A third construct was designed to contain a signalsequence, a FLAG epitope tag, the hinge region and CH2 and CH3 domainsof human IgG2, the transmembrane domain (TM) and intracellular domain(ICD) of human CD4, and green fluorescent protein (GFP) at theC-terminal end, referred to as Membrane-MAb(hIgG2)-GFP (FIG. 6A).

The portion of the hinge region used in each construct was designed toinclude the residues C-terminal of the cysteine involved in light chainpairing. The Membrane-MAb(mIgG1) construct was designed in the followingorder, signal sequence-FLAG-mIgG1 (hinge-CH2-CH3)-hCD4 (TM/ICD) and isshown in SEQ ID NO:24 (nucleotide sequence) and SEQ ID NO:22 (amino acidsequence with signal sequence). The Membrane-MAb(hIgG2) construct wasdesigned in the following order, signal sequence-FLAG-hIgG2(hinge-CH2-CH3)-hCD4 (TM/ICD) and is shown in SEQ ID NO:25 (nucleotidesequence) and SEQ ID NO:23 (amino acid sequence with signal sequence).The Membrane-MAb(hIgG2)-GFP construct was designed in the followingorder, signal sequence-FLAG-hIgG2 (hinge-CH2-CH3)-hCD4 (TM/ICD)-GFP andis shown in SEQ ID NO:27 (nucleotide sequence) and SEQ ID NO:26 (aminoacid sequence with signal sequence). The Membrane-MAb(hIgG2) andMembrane-Mab(hIgG2)-GFP constructs were designed to have a modificationwithin the CD4 intracellular domain to remove the lck protein bindingsite. The constructs were generated by chemical synthesis and clonedinto mammalian expression vector plasmids by standard techniques.

Example 2 Generation of Cell Line

The murine hybridoma fusion partner cell line SP2/0-Ag14 was stablytransfected with the Membrane-MAb(mIgG1) construct described inExample 1. 2×10⁶ SP2/0 cells were transfected with 2 ug of theMembrane-MAb(mIgG1) construct using an Amaxa® Nucleofection Kit V(Lonza) following the manufacturer's instructions for program L-013.After 24 hours, the cells were placed under 0.8 mg/ml G418 selection andpropagated in culture. Two weeks post-transfection, the cells werecharacterized by FACS analysis for cell surface expression of theMembrane-MAb(mIgG1) construct. These cells were designated SP2/0-MT.5×10⁶ transfected cells were incubated with fluorophore-labeledanti-FLAG antibody or an isotype negative control antibody (10 μg/ml)for 30 minutes on ice. The cells were washed, resuspended in DMEM/10%FBS, and were analyzed by flow cytometry. FIG. 2A shows the flowcytometry results with the negative control antibody (left panel) andthe number of cells expressing the Membrane-MAb(mIgG1) polypeptide asdetected with the anti-FLAG antibody (right panel).

Cells expressing the Membrane-MAb(mIgG1) construct were sorted by FACSat 1 cell per well into 96 well plates and grown in culture underselection with 0.5 mg/ml G418. After 10 days the cell clones from eachwell were analyzed for expression of the Membrane-MAb(mIgG1) constructby flow cytometry as described above and 5 sub-clones were selected. Asshown in FIG. 2B subclone SP2/0-MT.3 was observed to have the highestlevel of expression of the Membrane-MAb(mIgG1) construct.

Example 3 Use of the Membrane-MAb Technique to Isolate Hybridomas

Recombinant polypeptide fragments of the fri-domain of murine Frizzled 5(FZD5) and murine Frizzled 8 (FZD8) were generated for use as antigenfor antibody production. Standard recombinant DNA technology was used toisolate polynucleotides encoding amino acids 27-157 of FZD5 (SEQ IDNO:19) and amino acids 28-158 of FZD8 (SEQ ID NO:20). Thepolynucleotides were ligated in-frame N-terminal to a histidine-tag andcloned into a transfer plasmid vector for baculovirus-mediatedexpression in insect cells. Standard transfection, infection, and cellculture protocols were used to produce recombinant insect cellsexpressing the corresponding FZD polypeptides.

Mice (n=3) were immunized with both FZD5 and FZD8 antigen proteins usingstandard techniques. Blood from individual mice was screenedapproximately 70 days after initial immunization for antigen recognitionusing ELISA and FACS analysis. The two animals with the highest antibodytiters were selected for final antigen boost after which spleen cellswere isolated. The isolated splenocytes were fused with the SP2/0-MTcell line described in Example 2 using standard hybridoma fusiontechniques. The resulting hybridoma library was named 54L1.

Library 54L1 was screened with labeled FZD5 and labeled FZD8polypeptides. For labeled FZD5, murine His-tagged fri-domain FZD5 wasconjugated to Alexa Fluor™ 488 dye at a 15:1 dye:protein ratio followingthe manufacturer's protocol (In Vitrogen/Molecular Probes). For labeledFZD8, murine His-tagged fri-domain FZD8 was conjugated to Alexa Fluor™647 dye at a 15:1 dye:protein ratio following the manufacturer'sprotocol (InVitrogen/Molecular Probes), The hybridoma cells at 1×10⁷cells/ml were incubated with Alexa Fluor™ 488-labeled FZD5 (10 μg/ml)and Alexa Fluor 647-labeled FZD8 (10 μg/ml) for 30 minutes at roomtemperature. The cells were washed, resuspended in DMEM/10% FBS, andwere analyzed by flow cytometry (FIG. 3). Individual hybridoma cellsbound by both Alexa Fluor 488-labeled FZD5 and Alexa Fluor 647-labeledFZD8 were sorted by FACS at 1 cell per well into 96 well tissue cultureplates. As a control, individual cells from the 54L1 library wererandomly sorted at 1 cell per a well into 96 wells tissue cultureplates. The plates were incubated for 10 days to allow the hybridomacells to proliferate and the supernatant from each well was screened forthe presence of antibody that could bind both FZD5 and FZD8 protein.

For screening by FACS, HEK-293 cells were co-transfected with expressionvectors encoding a full-length cDNA clone of FZD5 or FZD8 and thetransfection marker GFP. 24 to 48 hours post-transfection, HEK-293 cellswere collected and incubated on ice with the anti-FZD5/8 hybridomasupernatants or control IgG. The cells were washed and bound primaryantibodies were detected with anti-mouse secondary antibodies conjugatedto a fluorescent chromophore. Labeled cells were then analyzed by FACSto identify antibodies that specifically recognized cell surfaceexpression of native FZD5 and/or FZD8.

The use of the SP2/0-MT fusion partner cell line and the Membrane-MAbtechnique resulted in selection of 526 out of 576 clones (91%) that werecapable of binding FZD5 and FZD8. In contrast, the control libraryscreening of random clones resulted in selection of only 11 out of 1705clones (0.6%) that were capable of binding FZD5 and FZD8 (see Table 1).Thus, use of the Membrane-MAb technique resulted in a dramatic increaseidentification of hybridomas specific for FZD5 and FZD8.

TABLE 1 Percent Cells FACS Positive/Total Clones Tested Positive RandomClones 11/1705 0.6% Sorted Clones 526/576   91%

As shown in FIG. 3, only a small percentage of the cells in library 54L1displayed binding to FZD5, FZD8, or both FZD5 and FZD8. The Membrane-MAbtechnique allowed for direct identification and selection of cellsproducing an antibody that bound to both FZD5 and FZD8. It should beclear to one of skill in the art that the cells producing antibodiesthat bind to only FZD5 or only FZD8 could also be selected by theMembrane-MAb technique.

Example 4 Use of the Membrane-MAb Technique to Isolate Hybridomas

A recombinant polypeptide fragment of the extracellular domain of humanDDR2 was generated for use as antigen for antibody production. Standardrecombinant DNA technology was used to isolate polynucleotides encodingamino acids 1-399 of DDR2 (SEQ ID NO:21). This polynucleotide wasligated in-frame N-terminal to a histidine-tag and cloned into atransfer plasmid vector for baculovirus-mediated expression in insectcells. Standard transfection, infection, and cell culture protocols wereused to produce recombinant insect cells expressing the correspondingDDR2 polypeptide.

Mice (n=3) were immunized with purified DDR2 antigen protein usingstandard techniques. Blood from individual mice was screenedapproximately 70 days after initial immunization for antigen recognitionusing ELISA and FACS analysis. The two animals with the highest antibodytiters were selected for final antigen boost after which spleen cellswere isolated and used to produce a DDR2 hybridoma library by standardhybridoma fusion techniques.

A portion of the DDR2 library was transfected with theMembrane-MAb(mIgG1) construct (described in Example 1). The library wastransfected using an Amaxa® Nucleofection Kit V (Lonza) following themanufacturer's instructions for program L-013. 3×10⁷ cells weretransfected with 30 μg of Membrane-Mab(mIgG1) construct (“Membrane-MAblibrary”). After 24 hours, the transfected cells were screened withlabeled DDR2 polypeptide. For labeled DDR2, DDR2 polypeptide (asprepared above) was conjugated to Alexa Fluor™ 488 carboxylic acidsuccinimidyl ester dye at a 15:1 dye:protein ratio following themanufacturer's protocol (InVitrogen/Molecular Probes). The Membrane-MAblibrary at 1×10⁷ cells/ml was incubated with Alexa Fluor 488-labeledDDR2 (20 m/ml) and PE-labeled anti-FLAG antibody (10 m/ml) for 30minutes on ice. The cells were washed, resuspended in DMEM/10% FBS, andwere analyzed by flow cytometry. As shown in FIG. 4 only a minorpercentage of the cells in the library displayed binding to DDR2 andanti-FLAG antibody.

Individual hybridoma cells bound by Alexa Fluor 488-labeled DDR2 andPE-labeled anti-FLAG antibody were sorted by FACS at 1 cell per wellinto 96 well tissue culture plates. Random individual cells from thesame library were deposited into wells of 96 wells tissue culture platesas a control. The plates were incubated for 10 days to allow thehybridoma cells to proliferate and subsequently, the supernatant fromeach well was screened for the presence of antibody that could bindfull-length DDR2 protein.

For screening by FACS, HEK-293 cells were co-transfected with expressionvectors encoding a full-length cDNA clone of DDR2 and the transfectionmarker GFP. 24 to 48 hours post-transfection, HEK-293 cells werecollected and incubated on ice with the anti-DDR2 hybridoma supernatantsor control IgG. The cells were washed and bound primary antibodies weredetected with anti-mouse secondary antibodies conjugated to afluorescent chromophore. Labeled cells were then analyzed by FACS toidentify antibodies that specifically recognized cell surface expressionof native DDR2.

The use of the Membrane-MAb technique resulted in selection of 141 outof 168 clones (84%) that were capable of binding DDR2. In contrast, thecontrol library screening of random clones resulted in selection of only16 out of 202 clones (8%) that were capable of binding DDR2 (see Table2). Thus, use of the Membrane MAb technique resulted in a dramaticincrease in identification of cells producing antigen specificantibodies.

TABLE 2 Percent Cells FACS Positive/Total Clones Tested Positive RandomClones  16/202  8% Sorted Clones 141/168 84%

Example 5 Generation of 293-hMT Cell Line

HEK-293 cells were stably transfected with the Membrane-MAb(hIgG2)-GFPconstruct to generate a cell line stably expressing theMembrane-MAb(hIgG2)-GFP polypeptide (described in Example 1 and depictedin FIG. 6C). 5×10⁶ HEK-293 cells were transfected with Bug of theMembrane-MAb(hIgG2)-GFP construct using the FuGENE transfection reagent(Roche, Indianapolis, Ind.) following the manufacturer's instructions.After 24 hours, the cells were placed under 0.8 mg/ml G418 selection andpropagated in culture. Two weeks post-transfection, the cells werecharacterized by FACS analysis for expression of theMembrane-MAb(hIgG2)-GFP construct. Subclones of the transfected cellswere screened for GFP expression and FIG. 5 shows the flow cytometryresults of ten clones.

Example 6 Single Gene Antibody Construct and Library

A parental antibody scaffold was designed as a single protein or singlechain antibody (scAb) construct in which the carboxyl terminus of theconstant region of the antibody light chain is linked to the aminoterminus of the heavy chain variable region via a (GGGGS)₆ peptidelinker. Cysteine residues in the hinge region were retained to allow fordisulfide linkages between two heavy chains or between one heavy chainand a Membrane-MAb construct. The light chain of the single geneantibody encodes a variable region and a human constant region. Theheavy chain of the single gene antibody encodes a variable region andhuman IgG CH1, CH2 and CH3 domains. An expression vector was constructedto produce the single chain antibody and referred to herein as MAbLibconstruct. The vector was designed so that the light chain variableregion and the heavy chain variable region are each flanked by uniquerestriction sites that allow for removal of the light chain variableregion and/or the heavy chain variable region and insertion of new anddifferent variable regions (as depicted in FIG. 6B). The MAbLibconstruct can be used to generate a wide variety of single chainantibody (scAb) libraries.

Light chain and heavy chain variable regions were PCR amplified fromhuman cDNA by methods well-known to those of skill in the art. Primerscontaining the restriction sites EcoRv or BsiWI were used in PCRreactions for light chain variable regions. Primers containing therestriction sites MfeI or BlpI were used in PCR reactions for heavychain variable regions. PCR products containing light chain variableregions were purified, digested with EcoRv and BsiWI and cloned into asimilarly digested parental MAbLib vector (described above). Thiscreated a scAb library with diversity in the light chain variableregion. Subsequently, PCR products containing heavy chain variableregions were purified, digested with MfeI and BlpI and cloned into asimilarly digested scAb library containing the diverse light chainvariable region. This created a scAb library with diversity in both thelight chain and heavy chain variable regions. One of skill in the artwould know that the heavy chain variable region may be inserted into theparental MAbLib vector prior to insertion of the light chain variableregion.

Example 7 Expression of Heterodimeric Antibody Molecule on Surface ofHEK-293 Cells

To validate formation of a heterodimeric antibody molecule on thesurface of cells, HEK-293 cells was transfected with DNA encoding ananti-DLL4 single chain antibody and DNA encoding theMembrane-MAb(hIgG2)-GFP protein. A synthetic polynucleotide encoding the21M18 light chain, a (GGGGS)₆ linker, the 21M18 variable heavy chain andCH1 domain was designed. The polynucleotide was synthesized by DNA2.0(Menlo Park Calif.) and cloned into MAbLib to generate a vector encodinga single chain 21M18 antibody (sc21M18) as described above in Example 6(sc21M18 SEQ ID NO:33, nucleotide sequence; SEQ ID NO:34, amino acidsequence). Anti-hDLL4 antibody 21M18 has been previously described inU.S. Pat. No. 7,750,124. sc21M18 plasmid DNA was transfected across arange of concentrations (25, 250, 2500, and 25000 ng/ml) and cells wereharvested after 48 hours. As controls, cells were transfected with thesc21M18 DNA only (-▴-), with the Membrane-MAb(hIgG2)-GFP DNA only (-▪-),or not transfected (-♦-). For detection of a functional anti-DLL4antibody molecule on the cell surface, transfected cells were incubatedwith an hDLL4-rFc fusion protein. Bound hDLL4-rFc was detected with aPE-labeled anti-rabbit Fc antibody. Cells were analyzed by FACS and meanfluorescence intensities (WI) of the cell populations were determined.As shown in FIG. 7, only cells transfected with the anti-DLL4 sc21M18DNA and the Membrane-MAb(hIgG2) DNA (—X—) expressed a membrane boundmolecule with a functional binding site that specifically bound theDLL4-rFc protein.

Example 8 Use of Carrier Plasmid to Modulate the Display of AntibodyMolecule

Transient transfection of mammalian cells with a plasmid generallyresults in multiple copies of plasmid per cell. The copy number per cellcan range from 100-10,000. The absolute number per cell depends on avariety of factors including, but not limited to, transfection protocol,transfection reagents, plasmid quality, plasmid size, and cellulardensity. In some embodiments, in generating libraries of cellsurface-displayed antibodies it is desirable to modulate the amount ofantibody-encoding plasmid (or Ab library plasmid) introduced into thecell. To modulate the plasmid copy number of Ab library plasmid incells, a separate “carrier” plasmid is used. The carrier plasmid ismixed with the Ab library plasmid and takes up some of the available“plasmid space”. To modulate the number of Ab library plasmid taken upby the cells, carrier plasmid is mixed with the Ab library plasmid atdifferent ratios and then transfected into cells.

The effect of carrier plasmid on the transfection and display of anAb-encoding plasmid was tested. The single chain anti-DLL4 antibody(sc21M18) described above was transfected with varying ratios of carrierplasmid. In this study, the carrier plasmid encoded an irrelevant singleprotein antibody (sc18R5). The cells were also transfected with theMembrane-MAb(hIgG2)-GFP construct. The concentration of Ab libraryplasmid in the cells could be inferred from the level of surfaceexpression of the anti-DLL4 antibody molecule. Approximately 5×10⁶HEK-293 cells were transfected with plasmid DNA and harvested after 48hours. The plasmid DNA was a mixture of sc21M18 and sc18R5, wherein theratio of sc21M18 to sc18R5 varied from 1 to 10,000-fold excess of sc18R5(1, 10, 100, 1000 or 10,000-fold excess). For detection of a functionalanti-DLL4 antibody molecule on the cell surface, transfected cells wereincubated with an hDLL4-rFc fusion protein. Bound hDLL4-rFc was detectedwith a PE-labeled anti-rabbit Fc antibody. The percentage of cells whichexpressed anti-DLL4 antibody was determined by FACS analysis. Cells werealso incubated with a control antigen (hJag-rFc, -▪-) or only thePE-labeled anti-rabbit Fc antibody (-▴-). As shown in FIG. 8, the amountof carrier plasmid DNA had a clear effect on the percentage of cellsexpressing anti-DLL4 antibody on the surface of transfected cells.

Example 9 Method of Screening Antibody Library

A study was performed using anti-DLL4 antibody sc21M18 plasmid DNA tovalidate the Membrane-Mab technique and selection methods. sc21M18plasmid DNA was mixed with irrelevant antibody sc18R5 plasmid DNA, wherethe sc18R5DNA was in 100.000-fold excess. HEK-293 cells were transfectedwith 7.5×10⁻⁶ μg sc21M18 plasmid DNA, 0.75 μg sc18R5 plasmid DNA, 14.3μg of carrier plasmid (kanamycin plasmid), and 15 μg of a plasmidencoding Membrane-MAb(hIgG2)-GFP. Cells were harvested after 48 hours.For detection of a functional anti-DLL4 antibody on the cell surface,transfected cells were incubated with an hDLL4-rFc fusion protein. BoundhDLL4-rFc was detected with a PE-labeled anti-rabbit Fc antibody.Labeled cells were analyzed and sorted by FACS to identify and isolateanti-DLL4 antibody expressing cells. hJag-rFc was used as a control, asthis antigen will not be recognized by the anti-DLL4 antibody. PlasmidDNA from FACS-sorted cells was isolated and amplified in bacteria. Theamplified plasmid DNA (in combination with the carrier plasmid andplasmid encoding Membrane-MAb(hIgG2)-GFP) was used to transfect freshHEK-293 cells and another round of selection was performed. This processof amplification and selection was iterated for 4 rounds and FACSresults are shown in FIG. 9. Table 3 shows the percentage of cellspositive for expression of anti-DLL4 antibody after each round ofselection. This study demonstrated that cells expressing anti-DLL4antibody were identified and enriched over 4 rounds of selection,despite the fact that the sc21M18 DNA had been highly diluted in abackground of irrelevant antibody DNA.

TABLE 3 Plasmid Concentration (μg) sc21M18 % Anti-DLL4 or AntibodyPositive Cells previous hDLL4- Round round sc18R5 Kanamycin MAb(hIgG2)rFc hJag1-rFc No antigen 1 7.5 × 10⁻⁶ 0.75 14.3 15 0.1 0.23 ND 2 0.75 015 15 0.23 0.78 0.002  Round 1 3 0.75 0 15 15 0.9 0.08 ND Round 2 4a0.75 0 15 15 2.9 0.14 0.08  Round 3 4b 7.0  0 0 0 32.8 0.43 0.0012 Round3

Example 10 Generation of an Anti-VEGF Antibody Library and Transfectioninto 293-hMT Cell Line

An antibody library was generated using a MAbLib construct expressingsc21M18 described above in Examples 6 and 7 and immunoglobulin cDNA frommice immunized with human vascular endothelial cell growth factor(hVEGF). Three Balb/c mice were immunized by intraperitoneal injectionof hVEGF. The initial injection contained hVEGF in Complete Freund'sadjuvant. Subsequent injections contained hVEGF in Incomplete Freund'sadjuvant. A total of four intraperitoneal injections were performed overthe course of three months. Mice received a final injection of hVEGF inPBS by intravenous tail vein injection. One week after the finalinjection spleens and lymph nodes were collected from the immunizedmice. RNA was isolated and cDNA was generated. DNA encoding murine heavychain variable regions was amplified by PCR, isolated, and purified. ThePCR products were digested with MfeI and BlpI and cloned into asimilarly digested sc21M18 construct, thereby replacing the heavy chainvariable region of SEQ ID NO:33, with a plurality of murine heavy chainvariable regions from hVEGF immunized mice. For this library, the 21M18light chain was held constant.

A second VEGF library was constructed similar to the library describedabove with the exception that the 21M18 light chain was replaced with aplurality of human kappa chain variable regions. Human kappa chainvariable regions were PCR amplified from pooled human cDNA using humankappa chain specific primers. The PCR products were isolated, purified,and digested with EcoRV and BsiWI and cloned into similarly digestedplasmid DNA from the first VEGF library, thereby replacing the 21M18light chain variable region.

293-hMT cells were transfected with the two anti-VEGF antibodylibraries.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included with the spirit and purview of this application.

All publications, patents and patent applications cited herein arehereby incorporated by reference in their entirety for all purposes tothe same extent as if each individual publication, patent or patentapplication were specifically and individually indicated to be soincorporated by reference.

SEQUENCES SEQ ID NO: 1 Mouse IgG1 constant region Nucleotide sequenceGGTTGTAAGCCTTGCATATGTACAGTCCCAGAAGTATCATCTGTCTTCATCTTCCCCCCAAAGCCCAAGGATGTGCTCACCATTACTCTGACTCCTAAGGTCACGTGTGTTGTGGTAGACATCAGCAAGGATGATCCCGAGGTCCAGTTCAGCTGGTTTGTAGATGATGTGGAGGTGCACACAGCTCAGACGCAACCCCGGGAGGAGCAGTTCAACAGCACTTTCCGCTCAGTCAGTGAACTTCCCATCATGCACCAGGACTGGCTCAATGGCAAGGAGTTCAAATGCAGGGTCAACAGTGCAGCTTTCCCTGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGCAGACCGAAGGCTCCACAGGTGTACACCATTCCACCTCCCAAGGAGCAGATGGCCAAGGATAAAGTCAGTCTGACCTGCATGATAACAGACTTCTTCCCTGAAGACATTACTGTGGAGTGGCAGTGGAATGGGCAGCCAGCGGAGAACTACAAGAACACTCAGCCCATCATGGACACAGATGGCTCTTACTTCGTCTACAGCAAGCTCAATGTGCAGAAGAGCAACTGGGAGGCAGGAAATACTTTCACCTGCTCTGTGTTACATGAGGGCCTGCACAACCACCATACTGAGAAGAGCCTCTCCCACTCTCCT GGTAAASEQ ID NO: 2 Mouse IgG1 constant region Amino acid sequenceGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGKSEQ ID NO: 3 Mouse IgG2a constant region Nucleotide sequenceATCAAGCCCTGTCCTCCATGCAAATGCCCAGCACCTAACCTCTTGGGTGGACCATCCGTCTTCATCTTCCCTCCAAAGATCAAGGATGTACTCATGATCTCCCTGAGCCCCATAGTCACATGTGTGGTGGTGGATGTGAGCGAGGATGACCCAGATGTCCAGATCAGCTGGTTTGTGAACAACGTGGAAGTACACACAGCTCAGACACAAACCCATAGAGAGGATTACAACAGTACTCTCCGGGTGGTCAGTGCCCTCCCCATCCAGCACCAGGACTGGATGAGTGGCAAGGAGTTCAAATGCAAGGTCAACAACAAAGACCTGCCAGCGCCCATCGAGAGAACCATCTCAAAACCCAAAGGGTCAGTAAGAGCTCCACAGGTATATGTCTTGCCTCCACCAGAAGAAGAGATGACTAAGAAACAGGTCACTCTGACCTGCATGGTCACAGACTTCATGCCTGAAGACATTTACGTGGAGTGGACCAACAACGGGAAAACAGAGCTAAACTACAAGAACACTGAACCAGTCCTGGACTCTGATGGTTCTTACTTCATGTACAGCAAGCTGAGAGTGGAAAAGAAGAACTGGGTGGAAAGAAATAGCTACTCCTGTTCAGTGGTCCACGAGGGTCTGCACAATCACCACACGACTAAGAGCTTCTCCCGGACTCCGGGTAAASEQ ID NO: 4 Mouse IgG2a constant region Amino acid sequenceIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGKSEQ ID NO: 5 Human IgG1 constant region Nucleotide sequenceACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAASEQ ID NO: 6 Human IgG1 constant region Amino acid sequenceTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO: 7 Human IgG2 constant region Nucleotide sequenceTGTGTCGAGTGCCCACCTTGCCCAGCACCACCTGTGGCAGGACCTTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCCGAGGTCCAGTTTAATTGGTATGTCGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCACGGGAGGAGCAGTTCAACAGCACATTCAGGGTGGTCAGCGTCCTCACCGTTGTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGTCCAACAAAGGCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGGCAGCCCAGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTGAAGGGATTTTATCCTTCCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCTGAGAACAACTACAAGACCACACCTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTGTATTCCAAACTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTG TCCCCTGGASEQ ID NO: 8 Human IgG2 constant region Amino acid sequenceCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSEQ ID NO: 9 mIgG1-hCD4 const uct without signal sequence andwithout FLAG tag Nucleotide sequenceGCGATCGCGGGTTGTAAGCCTTGCATATGTACAGTCCCAGAAGTATCATCTGTCTTCATCTTCCCCCCAAAGCCCAAGGATGTGCTCACCATTACTCTGACTCCTAAGGTCACGTGTGTTGTGGTAGACATCAGCAAGGATGATCCCGAGGTCCAGTTCAGCTGGTTTGTAGATGATGTGGAGGTGCACACAGCTCAGACGCAACCCCGGGAGGAGCAGTTCAACAGCACTTTCCGCTCAGTCAGTGAACTTCCCATCATGCACCAGGACTGGCTCAATGGCAAGGAGTTCAAATGCAGGGTCAACAGTGCAGCTTTCCCTGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGCAGACCGAAGGCTCCACAGGTGTACACCATTCCACCTCCCAAGGAGCAGATGGCCAAGGATAAAGTCAGTCTGACCTGCATGATAACAGACTTCTTCCCTGAAGACATTACTGTGGAGTGGCAGTGGAATGGGCAGCCAGCGGAGAACTACAAGAACACTCAGCCCATCATGGACACAGATGGCTCTTACTTCGTCTACAGCAAGCTCAATGTGCAGAAGAGCAACTGGGAGGCAGGAAATACTTTCACCTGCTCTGTGTTACATGAGGGCCTGCACAACCACCATACTGAGAAGAGCCTCTCCCACTCTCCTGGTAAAGGGCGCGCCATGGCCCTGATTGTGCTGGGGGGCGTCGCCGGCCTCCTGCTTTTCATTGGGCTAGGCATCTTCTTCTGTGTCAGGTGCCGGCACCGAAGGCGCCAAGCAGAGCGGATGTCTCAGATCAAGAGACTCCTCAGTGAGAAGAAGACCTGCCAGTGCCCTCACCGGTTTCAGAAGACATGTAGCCCCATTTAGSEQ ID NO: 10 mIgG1-hCD4 construct without signal sequence andwithout FLAG tag Amino acid sequenceAIAGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGKGRAMALIVLGGVAGLLLFIGLGIFECVRCRHRRRQAERMSQIKRLLSEKKTCQCPHRFQKTCSPISEQ ID NO: 11 hIgG2-hCD4* construct without signal sequence andwithout FLAG tag Nucleotide sequenceGCGATCGCGAACGGATGTGTCGAGTGCCCACCTTGCCCAGCACCACCTGTGGCAGGACCTTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCCGAGGTCCAGTTTAATTGGTATGTCGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCACGGGAGGAGCAGTTCAACAGCACATTCAGGGTGGTCAGCGTCCTCACCGTTGTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGTCCAACAAAGGCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGGCAGCCCAGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTGAAGGGATTTTATCCTTCCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCTGAGAACAACTACAAGACCACACCTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTGTATTCCAAACTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCCCCTGGAAAGGGGCGCGCCATGGCCCTGATTGTGCTGGGGGGCGTCGCCGGCCTCCTGCTTTTCATTGGGCTCGGCATCTTCTTCTGTGTCCGCTGCCGGCACCGACGCCGCCAAGCAGAGCGGATGTCTCAGATCAAGAGACTCCTCAGTGAGAAGAAGACCGCACAGTGCCCTCACCGGTTTCAGAAGACATGTAGCCCCATTTAGSEQ ID NO: 12 hIgG2-hCD4* construct without signal sequence andwithout FLAG ta gAmino acid sequenceAIANGCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGRAMALIVLGGVAGLLLFIGLGIFECVRCRHRRRQAERMSQIKRLLSEKKTAQCPHREQKTCSPI*CD4 Intracellular domain region is modified, amino acidchange is underlined SEQ ID NO: 13 Human CD4 TM sequenceMALIVLGGVAGLLLFIGLGIFF SEQ ID NO: 14 Human CD4 TM-ICD sequenceMALIVLGGVAGLLLFIGLGIFFCVRCRHRRRQAERMSQIKRLLSEKKTCQCPHRFQKTCSPISEQ ID NO: 15 Human CD4 TM-ICD*MALIVLGGVAGLLLFIGLGIFFCVRCRHRRRQAERMSQIKRLLSEKKTAQCPHRFQKTCSPI*CD4 Intracellular domain region is modified, amino acidchange is underlined SEQ ID NO: 16 Mouse CD4 TM sequenceFLACVLGGSFGFLGFLGLCILC SEQ ID NO: 17 Mouse CD4 TM-ICD sequenceFLACVLGGSFGFLGFLGLCILCCVRCRHQQRQAARMSQIKRLLSEKKTCQCPHRMQKSHNLISEQ ID NO: 18 FLAG tag DYKDDDDKSEQ ID NO: 19 Human FZD5 Fri-domain sequence (Amino acids 27-157)ASKAPVCQEITVPMCRGIGYNLTHMPNQFNHDTQDEAGLEVHQFWPLVEIQCSPDLRFFLCSMYTPICLPDYHKPLPPCRSVCERAKAGCSPLMRQYGFAWPERMSCDRLPVLGRDAEVL CMDYNRSEATTSEQ ID NO: 20 Human FZD8 Fri-domain sequence (Amino acids 28-158)ASAKELACQEITVPLCKGIGYNYTYMPNQFNHDTQDEAGLEVHQFWPLVEIQCSPDLKFFLCSMYTPICLEDYKKPLPPCRSVCERAKAGCAPLMRQYGFAWPDRMRCDRLPEQGNPDTL CMDYNRTDLTTSEQ ID NO: 21 Human DDR2 Amino acids 1-399MIL I PRMLLVLFLLLPILSSAKAQVNPAICRYPLGMSGGQIPDEDITASSQWSESTAAKYGRLDSEEGDGAWCPEIPVEPDDLKEFLQIDLHTLHFITLVGTQGRHAGGHGIEFAPMYKINYSRDGTRWISWRNRHGKQVLDGNSNPYDIFLKDLEPPIVARFVRFIPVTDHSMNVCMRVELYGCVWLDGLVSYNAPAGQQFVLPGGSIIYLNDSVYDGAVGYSMTEGLGQLTDGVSGLDDFTQTHEYHVWPGYDYVGWRNESATNGYIEIMFEFDRIRNFTTMKVECNNMFAKGVKIFKEVQCYFRSEASEWEPNAISFPLVLDDVNPSARFVTVPLHHRMASAIKCQYHFADTWMMFSEITFQSDAAMYNNSEALPTSPMAPTTYDPMLKVDDSNTRSEQ ID NO: 22 mIgG1-hCD4 construct with signal sequence and withFLAG tag Amino Acid sequenceMSALLILALVGAAVADYKDHDGDYKDHDIDYKDDDDKAIAGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGKGRAMALIVLGGVAGLLLFIGLGIFFCVRCRHRRRQAERMSQIKRLLSEKKTCQCPHRFQKTCSPISEQ ID NO: 23 hIgG2-hCD4* construct with signal sequence and withFLAG tag Amino Acid sequenceMSALLILALVGAAVADYKDHDGDYKDHDIDYKDDDDKAIANGCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGRAMALIVLGGVAGLLLFIGLGIFFCVRCRHRRRQAERMSQIKRLLSEKKTAQCPHRFQKTCSPI*CD4 Intracellular domain region is modified, amino acid changeis underlinedSEQ ID NO: 24 mIgG1-hCD4 construct with signal sequence and withFLAG tag Nucleotide sequenceATGTCTGCACTTCTGATCCTAGCTCTTGTTGGAGCTGCAGTTGCTGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGGCGATCGCGGGTTGTAAGCCTTGCATATGTACAGTCCCAGAAGTATCATCTGTCTTCATCTTCCCCCCAAAGCCCAAGGATGTGCTCACCATTACTCTGACTCCTAAGGTCACGTGTGTTGTGGTAGACATCAGCAAGGATGATCCCGAGGTCCAGTTCAGCTGGTTTGTAGATGATGTGGAGGTGCACACAGCTCAGACGCAACCCCGGGAGGAGCAGTTCAACAGCACTTTCCGCTCAGTCAGTGAACTTCCCATCATGCACCAGGACTGGCTCAATGGCAAGGAGTTCAAATGCAGGGTCAACAGTGCAGCTTTCCCTGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGCAGACCGAAGGCTCCACAGGTGTACACCATTCCACCTCCCAAGGAGCAGATGGCCAAGGATAAAGTCAGTCTGACCTGCATGATAACAGACTTCTTCCCTGAAGACATTACTGTGGAGTGGCAGTGGAATGGGCAGCCAGCGGAGAACTACAAGAACACTCAGCCCATCATGGACACAGATGGCTCTTACTTCGTCTACAGCAAGCTCAATGTGCAGAAGAGCAACTGGGAGGCAGGAAATACTTTCACCTGCTCTGTGTTACATGAGGGCCTGCACAACCACCATACTGAGAAGAGCCTCTCCCACTCTCCTGGTAAAGGGCGCGCCATGGCCCTGATTGTGCTGGGGGGCGTCGCCGGCCTCCTGCTTTTCATTGGGCTAGGCATCTTCTTCTGTGTCAGGTGCCGGCACCGAAGGCGCCAAGCAGAGCGGATGTCTCAGATCAAGAGACTCCTCAGTGAGAAGAAGACCTGCCAGTGCCCTCACCGGTTTCAGAAGACATGTAGCCCCATTTAGSEQ ID NO: 25 hIgG2-hCD4* construct with signal sequence and withFLAG tag Nucleotide sequenceATGTCTGCACTCCTGATCCTCGCTCTCGTTGGAGCTGCAGTTGCTGACTACAAAGACCATGACGGAGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGGCGATCGCGAACGGATGTGTCGAGTGCCCACCTTGCCCAGCACCACCTGTGGCAGGACCTTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCCGAGGTCCAGTTTAATTGGTATGTCGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCACGGGAGGAGCAGTTCAACAGCACATTCAGGGTGGTCAGCGTCCTCACCGTTGTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGTCCAACAAAGGCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGGCAGCCCAGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTGAAGGGATTTTATCCTTCCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCTGAGAACAACTACAAGACCACACCTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTGTATTCCAAACTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCCCCTGGAAAGGGGCGCGCCATGGCCCTGATTGTGCTGGGGGGCGTCGCCGGCCTCCTGCTTTTCATTGGGCTCGGCATCTTCTTCTGTGTCCGCTGCCGGCACCGACGCCGCCAAGCAGAGCGGATGTCTCAGATCAAGAGACTCCTCAGTGAGAAGAAGACCGCACAGTGCCCTCACCGGTTTCAGAAGACATGTAGCCCCATTTAGSEQ ID NO: 26 hIgG2-hCD4-GFP construct with signal sequence andwith FLAG tag Amino Acid sequenceMSALLILALVGAAVADYKDHDGDYKDHDIDYKDDDDKAIANGCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGRAMALIVLGGVAGLLLFIGLGIFFCVRCRHRRRQAERMSQIKRLLSEKKTAQCPHRFQKTCSPIMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHKVYITADKQKNGIKVNFKTRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKSEQ ID NO: 27 hIgG2-hCD4-GFP construct with signal sequence andwith FLAG tag Nucleotide sequenceATGTCTGCACTCCTGATCCTCGCTCTCGTTGGAGCTGCAGTTGCTGACTACAAAGACCATGACGGAGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGGCGATCGCGAACGGATGTGTCGAGTGCCCACCTTGCCCAGCACCACCTGTGGCAGGACCTTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCCGAGGTCCAGTTTAATTGGTATGTCGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCACGGGAGGAGCAGTTCAACAGCACATTCAGGGTGGTCAGCGTCCTCACCGTTGTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGTCCAACAAAGGCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGGCAGCCCAGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTGAAGGGATTTTATCCTTCCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCTGAGAACAACTACAAGACCACACCTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTGTATTCCAAACTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCCCCTGGAAAGGGGCGCGCCATGGCCCTGATTGTGCTGGGGGGCGTCGCCGGCCTCCTGCTTTTCATTGGGCTCGGCATCTTCTTCTGTGTCCGCTGCCGGCACCGACGCCGCCAAGCAGAGCGGATGTCTCAGATCAAGAGACTCCTCAGTGAGAAGAAGACCGCACAGTGCCCTCACCGGTTTCAGAAGACATGTAGCCCCATTATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCTTGACCTACGGCGTGCAGTGCTTCGCCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAAGGTCTATATCACCGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGACCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAGTAGSEQ ID NO: 28 hIgG2-hCD4-GFP construct without signal sequenceand without FLAG tag Amino Acid sequenceCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGRAMALIVLGGVAGLLLFIGLGIFFCVRCRHRRRQAERMSQIKRLLSEKKTAQCPHRFQKTCSPIMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHKVYITADKQKNGIKVNFKTRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKSEQ ID NO: 29 hIgG2-hCD4-GFP construct without signal sequenceand without FLAG tag Nucleotide sequenceTGTGTCGAGTGCCCACCTTGCCCAGCACCACCTGTGGCAGGACCTTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCCGAGGTCCAGTTTAATTGGTATGTCGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCACGGGAGGAGCAGTTCAACAGCACATTCAGGGTGGTCAGCGTCCTCACCGTTGTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGTCCAACAAAGGCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGGCAGCCCAGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTGAAGGGATTTTATCCTTCCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCTGAGAACAACTACAAGACCACACCTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTGTATTCCAAACTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCCCCTGGAAAGGGGCGCGCCATGGCCCTGATTGTGCTGGGGGGCGTCGCCGGCCTCCTGCTTTTCATTGGGCTCGGCATCTTCTTCTGTGTCCGCTGCCGGCACCGACGCCGCCAAGCAGAGCGGATGTCTCAGATCAAGAGACTCCTCAGTGAGAAGAAGACCGCACAGTGCCCTCACCGGTTTCAGAAGACATGTAGCCCCATTATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCTTGACCTACGGCGTGCAGTGCTTCGCCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAAGGTCTATATCACCGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGACCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAGTAGSEQ ID NO: 30 hIgG2-hCD4-GFP construct - predicted mature proteinAmino Acid sequenceDYKDHDGDYKDHDIDYKDDDDKAIANGCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGRAMALIVLGGVAGLLLFIGLGIFFCVRCRHRRRQAERMSQIKRLLSEKKTAQCPHRFQKTCSPIMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHKVYITADKQKNGIKVNFKTRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKSEQ ID NO: 31 mIgG1-hCD4 construct - predicted mature proteinAmino Acid sequenceDYKDHDGDYKDHDIDYKDDDDKAIAGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDITSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGKGRAMALIVLGGVAGLLLFIGLGIFFCVRCRHRRRQAERMSQIKRLLSEKKTCQCPHRFQKTCSPISEQ ID NO: 32 hIgG2-hCD4 construct - predicted mature proteinAmino Acid sequenceDYKDHDGDYKDHDIDYKDDDDKAIANGCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGRAMALIVLGGVAGLLLFIGLGIFFCVRCRHRRRQAERMSQIKRLLSEKKTAQCPHRFQKTCSPISEQ ID NO: 33 MAbLib construct (sc21M18 sequence) predicted signalsequence underlined Nucleotide sequenceATCGTACTCCAAACCCAAGTATTCATCTCGCTGCTGTTGTGGATTAGCGGAGCCTATGGAGATATCGTCATGACACAGTCACCGGACTCGCTTGCAGTATCGCTCGGCGAGAGGGCCACCATTTCGTGTCGCGCTTCAGAGTCAGTCGATAACTACCGGATCTCGTTTATGAAGTGCTTTCAGCAGAAGCCCGGACAACCACCGAAGTTGCTCATCTACGCGGCTTCAAATCAGGGGTCAGGGGTCCCTGACAGATTTTCCGGCTCCGGTTCCGGTACAGATTTCACGCTGACCATCTCGTCGCTGCAGGCCGAGGACGTGGCCGTGTATTACTGCCAGCAGTCAAAAGAGGTCCCCTGGACTTTTGGGGGTGGGACGAAAGTGGAGATCAAGCGTACGGTGGCGGCACCTTCAGTGTTTATCTTCCCGCCGTCCGACGAACAGCTTAAGTCCGGTACGGCGTCGGTAGTCTGCCTGCTGAACAATTTCTATCCCAGGGAAGCGAAAGTACAATGGAAGGTCGACAATGCCCTCCAGAGCGGGAATAGCCAAGAATCGGTCACAGAACAGGATTCGAAGGACTCAACGTATAGCCTTTCGTCCACACTTACACTCTCGAAAGCTGACTATGAGAAGCATAAGGTCTATGCATGTGAAGTCACTCATCAAGGTCTTTCGTCGCCCGTAACCAAGAGCTTCAACCGCGGAGAGTGTGGAGGAGGTGGTGGATCAGGCGGTGGTGGGTCGGGAGGGGGTGGCAGCGGAGGAGGGGGATCCGGTGGAGGGGGTAGCGGGGGAGGAGGGAGCCAGGTGCAATTGGTGCAGTCCGGGGCAGAAGTGAAGAAGCCTGGCGCGTCAGTGAAGATCAGCTGCAAAGCCTCGGGGTATTCCTTTACAGCATACTACATTCACTGGGTCAAACAGGCGCCAGGACAGGGGTTGGAGTGGATTGGATACATTTCCTCGTACAACGGGGCCACGAACTACAATCAGAAATTCAAAGGACGGGTGACGTTTACTACGGACACCAGCACTTCGACGGCGTACATGGAGCTTCGATCACTCCGGTCCGATGACACGGCTGTATACTACTGTGCCAGAGATTATGATTATGATGTGGGAATGGACTACTGGGGACAGGGGACATTGGTAACAGTGAGCTCAGCCAGCACAAAGGGCCCTAGCGTCTTCCCTCTGGCCCCCTGCAGCAGGAGCACCAGCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCTCTGACCAGCGGCGTGCACACCTTCCCAGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAACTTCGGCACCCAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGACAGTTGAGCGCAAATGTTGTGTCGAGTGCCCACCGTGCCCAGCACCACCTGTGGCAGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCACGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCACGGGAGGAGCAGTTCAACAGCACGTTCCGTGTGGTCAGCGTCCTCACCGTTGTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACACCTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAASEQ ID NO: 34 MAbLib construct (sc21M18 sequence) predicted signalsequence underlined Amino Acid sequenceMVLQTQVFISLLLWISGAYGDIVMTQSPDSLAVSLGERATISCRASESVDNYGISFMKWFQQKPGQPPKLLIYAASNQGSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSKEVPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKISCKASGYSFTAYYIHWVKQAPGQGLEWIGYISSYNGATNYNQKFKGRVTFTTDTSTSTAYMELRSLRSDDTAVYYCARDYDYDVGMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

What is claimed:
 1. A method of producing a cell library expressingheterodimeric molecules on the cell surface, comprising: (a) fusing afirst population of cells that express a first polypeptide with a secondpopulation of cells that comprise a plurality of polynucleotides thatencode for a plurality of second polypeptides; wherein the firstpolypeptide comprises (i) an extracellular portion comprising animmunoglobulin heavy chain constant region comprising CH2 and CH3domains; and (ii) a non-immunoglobulin transmembrane portion, whereinthe first polypeptide comprises a sequence at least 80% identical to SEQID NO:10, SEQ ID NO:12, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:26, SEQ IDNO:28, SEQ ID NO:30, SEQ ID NO:31, or SEQ ID NO:32; and wherein thesecond polypeptide comprises an immunoglobulin heavy chain constantregion comprising CH2 and CH3 domains; (b) incubating the fused cells toallow expression of heterodimeric molecules on the surface of the cells,wherein the heterodimeric molecules comprise the first polypeptide andone of the second polypeptides.
 2. The method of claim 1, comprisingtransfecting the first population of cells with a polynucleotide thatencodes the first polypeptide.
 3. The method of claim 2, wherein thepolynucleotide encoding the first polypeptide is transiently transfectedor stably transfected into the population of cells.
 4. The method ofclaim 1, wherein the first population of cells is a fusion partner cellline.
 5. The method of claim 1, wherein the second population of cellscomprise antibody-producing cells.
 6. The method of claim 5, wherein theantibody-producing cell is selected from the group consisting of aB-cell, a plasma cell, a hybridoma, a myeloma, and a recombinant cell.7. The method of claim 5, wherein the antibody-producing cell is from anaive animal or from an immunized animal.
 8. The method of claim 5,wherein the antibody-producing cell is a mouse cell or a human cell. 9.The method of claim 1, wherein the plurality of polynucleotides of thesecond population of cells comprises a DNA library.
 10. The method ofclaim 1, wherein the first polypeptide comprises a sequence at least 80%identical to SEQ ID NO:12.
 11. The method of claim 1, wherein the secondpolypeptide is associated with an immunoglobulin light chain and formsan antigen-binding site.
 12. The method of claim 1, wherein the firstpolypeptide forms at least one disulfide bond with the secondpolypeptide to form the heterodimeric molecule.
 13. The method of claim1, wherein the heterodimeric molecule comprises one antigen-bindingsite.
 14. The method of claim 1, wherein the cell library comprisesmammalian cells.
 15. The method of claim 1, further comprising: (c)contacting the fused cells with a detection molecule, wherein thedetection molecule binds the heterodimeric molecule.
 16. The method ofclaim 15, wherein the detection molecule is labeled.
 17. The method ofclaim 15, further comprising: (d) identifying the fused cells bound bythe detection molecule.
 18. The method of claim 17, wherein identifyingthe fused cells bound by the detection molecule comprises using flowcytometry or fluorescence-activated cell sorting (FACS).
 19. The methodof claim 17, further comprising: (e) isolating the fused cells bound bythe detection molecule.